ИСТИНА

Cell death is a central feature of aging, however there have been no systematic data on cellular systems whose perturbation increases the chance of cell death. Many species, including humans, exhibit two life periods wherein their chance of death is high: the late life and early ages, but the causes of death in early life are not well understood. Yeast has been a useful model to study aging and, in this work, we used it to screen for genes whose deficiency increases the death rate of yeast cells, as well as to determine the age-specificity of this death. We used phloxine, a non-toxic die that does not penetrate cells with intact membranes, but effectively stains dead cells, and applied this assay to the full yeast knockout collection as well as a collection of strains with downregulated essential genes. This screen identified ~200 genes, about 2/3 of them being essential. We confirmed these results by staining with propidium iodide coupled with flow cytometry. Interestingly, phloxine staining of colonies revealed different patterns of cell death – some mutants were more prone to die at the edge of the colony, others were stained uniformly, and others preferentially died at the colony center. This revealed different sensitivity of the mutants to their environment, such as contact with the medium, starvation, and high density growth. To determine the replicative age at which the cells were most likely to die, we developed the DIVision Arrest assay (DIVA), which involves staining of cell wall with a fluorescent agent, division of this population ~10-15 times, and quantification of budscars of the stained cells. DIVA allows detection of cells that stopped dividing at early replicative ages, as compared to the majority of stained cells. An increased chance of division arrest at replicative age zero was observed in a considerable number of the phloxine positive strains (~20%). Interestingly, we also observed an early-age division arrest in some phloxine-negative strains which were randomly selected from strains with downregulated essential genes (~5%). Our data represent the first systematic study of genes whose deficiency increased the chance of early cell death. It also identifies distinct death patterns in yeast mutants, suggesting different mechanisms of cell death, and demonstrates division arrest at early ages in a large number of strains. This work was supported by the Russian Federation grant 14.W03.31.0012 and the Ministry of Science and Higher Education of the Russian Federation.