ИСТИНА

Cell death and its deregulation are central features of aging, cancer and various degenerative diseases in humans. Understanding cell death in microorganisms is also highly relevant for the development of drugs as well as for a basic understanding of how living systems function and break down. In this work, we have used Saccharomyces cerevisiae to screen for genetic perturbations that increase the death rate of yeast cells on rich medium with glucose as a carbon source. Using phloxine, a non-toxic die that does not penetrate cells with intact membranes, but effectively stains dead cells, we assayed the full yeast knockout collection as well as a collection of strains with downregulated essential genes. This screen identified 127 genes, about 2/3 of them being essential. We confirmed these results by flow cytometry of propidium iodide-stained cells. 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 suggests 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 walls with a fluorescent agent, doubling of the culture ~10 times in order to age the stained cells, 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. This assay, together with several others, showed that a considerable number of phloxine-positive mutants exhibited rapid cell permeabilization at early replicative age. Interestingly, we also observed an early-age division arrest in some phloxine-negative strains which were randomly selected from strains with downregulated essential genes. 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, Grant from the President of the Russian Federation for Young Scientists 075-15-2019-598 and the Ministry of Science and Higher Education of the Russian Federation.