Gene-dependent cell death in yeast

X Teng,M D Boersma,F J Pineda,W-C Cheng,B Qi,K Ramachandran,P V Lehmann,T-X Yu,J M Hardwick,T Hattier

doi:10.1038/cddis.2011.72

Abstract

Caspase-dependent apoptotic cell death has been extensively studied in cultured cells and during embryonic development, but the existence of analogous molecular pathways in single-cell species is uncertain. This has reduced enthusiasm for applying the advanced genetic tools available for yeast to study cell death regulation. However, partial characterization in mammals of additional genetically encoded cell death mechanisms, which lead to a range of dying cell morphologies and necrosis, suggests potential applications for yeast genetics. In this light, we revisited the topic of gene-dependent cell death in yeast to determine the prevalence of yeast genes with the capacity to contribute to cell-autonomous death. We developed a rigorous strategy by allowing sufficient time for gene-dependent events to occur, but insufficient time to evolve new populations, and applied this strategy to the Saccharomyces cerevisiae gene knockout collection. Unlike sudden heat shock, a ramped heat stimulus delivered over several minutes with a thermocycler, coupled with assessment of viability by automated counting of microscopic colonies revealed highly reproducible gene-specific survival phenotypes, which typically persist under alternative conditions. Unexpectedly, we identified over 800 yeast knockout strains that exhibit significantly increased survival following insult, implying that these genes can contribute to cell death. Although these death mechanisms are yet uncharacterized, this study facilitates further exploration.

Highlights

Before pursuing a strategy to uncover potential cell death genes in yeast, it is useful to articulate a generally acceptable definition of a cell death gene that applies to both single- and multi-cellular species
While performing the hundreds of trials required for developing treatment conditions that reliably detect genedependent yeast cell death, we routinely compared three test strains with distinct susceptibilities to cell death
We found that B2000 yeast knockout strains are more resistant to cell death than wild type, and B800 different gene knockouts are more resistant to death than the DNM1 (DRP1) knockout

Summary

Introduction

Before pursuing a strategy to uncover potential cell death genes in yeast, it is useful to articulate a generally acceptable definition of a cell death gene that applies to both single- and multi-cellular species. In this study we will adopt the term programmed cell death in the broadest sense, referring to any genetically encoded function of the dying cell that contributes importantly to its own demise This definition can be justified by the following arguments. It is not currently possible to rigorously determine if the pathological cell death during ischemic injury in adults is evolved, accidental or a combination thereof, because knockout animals could be expected to have the same phenotype in any case. This effort is further complicated if the pro-death gene has essential pro-survival functions (e.g., calpain). Because we found that the available yeast cell death assays were not amenable to this task, we first had to develop a strategy to reliably quantify gene-dependent cell death of yeast

Methods

Results

Conclusion