PART III CELL DEATH IN NONMAMMALIAN ORGANISMS

The yeast Saccharomyces cerevisiae is one of the most studied model systems for molecular and cellular biology. In 1996, it became the first eukaryotic organism to have a completely sequenced genome (Dujon, 1996; Goffeau et al., 1996), which led to a number of valuable and widely accessed databases. Among its features is the short generation time (usually 90–120 minutes) and the ability to grow at various temperatures in relatively inexpensive media. Moreover, many of its genes are well characterized, thanks in part to its amenability to modifications such as gene disruption, gene marking, mutations, or gene-dosage modifications. Because of these advantageous features, it has become the model organism of choice for many investigators in fields ranging from basic biology to biomedical research.

One of the most studied subjects of the past decade is the programmed cell death, or apoptosis, a highly coordinated cellular suicide program that is crucial for maintenance of health and tissue function and the focus of this book. Apoptosis is a very complex phenomenon that is relatively well understood and is implicated in diseases ranging from cancer to neurodegenerative disorders. Many of the apoptosis-related genes were discovered and studied in model organisms, primarily in Drosophila melanogaster and Caenorhabditis elegans. Because for the unicellular yeast programmed cell death implies a highly controversial organismal suicide, the research on yeast apoptosis started late. However, during the last 10 years, many studies reported on the existence of programmed cell death in yeast and, at present, at least the possibility that a single cell organism can undergo a death program is becoming widely accepted. It is still not yet widely accepted that yeast are capable of undergoing apoptosis as strictly defined in animal species, in part because these simple eukaryotes lack caspases. For purposes of this chapter, however, we nevertheless

refer to the apoptosis-like cell death process of yeast as apoptosis.

Apoptosis in yeast can be induced by a variety of compounds and conditions, including hydrogen peroxide and acetic acid, amiodarone, hyperosmotic stress, and aging. Genetics studies also contributed to the understanding of the mechanisms of cell death in yeast by revealing the role of various genes in a form of cell death accompanied by the appearance of features of mammalian apoptotic cells. Some of these mutations are in analogues of crucial components of the apoptotic cascade in mammals such as yeast apoptosis-inducing factor (AIF), metacaspase (YCA1), an inhibitor of apoptosis protein (BIR1), OMI/Htr2A (nuclear mediator of apoptosis; NMA111), and DJ-1 (HSP31), as well as a nuclease (TAT-D) that is apparently involved in DNA degradation during apoptosis.

Likewise, some genes involved in yeast cell death have been confirmed as apoptotic regulators in metazoans. Although the yeast genome does not appear to contain obvious orthologs of the mammalian Bax and Bcl family genes, it was shown that cell death in S. cerevisiae can be induced by the expression of Bax, and it is accompanied by typical features of apoptosis, such as externalization of phosphatidylserine at the surface of the cytoplasmic membrane, cytochrome c release, membrane blebbing, chromatin condensation and margination, and DNA cleavage. The simultaneous expression of Bcl-xL or Bcl-2 prevents these effects and cell death. More importantly, Bax-mediated cell death in yeast, as in mammalian cells, involves mitochondrial dysfunction, leading to the release of cyt c and apoptosis, supporting the hypothesis that Bax can function in yeast in a way analogous to its role in mammals. Therefore, mitochondria play a central role in both metazoan and yeast apoptosis. In fact, in addition to cytochrome c,

mitochondria represent the home of many proapoptotic molecules (i.e., AIF, endonuclease G) and are the site of early morphological changes that occur during programmed cell death. Fragmentation of tubular mitochondrial network is visible in yeast cells treated with acetic acid, H2O2, amiodarone, or ethanol. These changes are similar to the thread-to-grain transition observed in mammalian mitochondria during apoptosis. These similarities between the mammalian and yeast apoptotic pathways are represented in Figure 33-1.

1. PHENOTYPE AND ASSAYS OF YEAST APOPTOSIS

Various techniques, such as dyes based on metabolic activity including MTT or phloxine B, are available to measure the viability of yeast cells (Cannon et al., 1986; Teparic et al., 2004), but the counting of colonies generated by viable individual cells is the simplest and preferred method. In fact, in contrast to cultured mammalian cells, viable individual yeast cells reliably give rise to colonies. A defined number of viable cells (usually <500) are plated on complete media, and resulting colonies are counted after 2 to 3 days of incubation. In the basic clonogenic assay, cells are counted in a Thoma chamber at the microscope or by counting the cells directly on the plate. Alternatively, a cellular suspension can be deposited onto a 1-mm-thick pad of rich medium

with 2% agarose on a microscope slide, and after the incubation of slides for approximately 18 hours, the percentage of alive/dead cells is determined based on the ratio of plated cells that are able to form a micro-colony. As for mammalian cells, DNA fragmentation in yeast can be measured with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, although before the attachment to slides and subsequent staining, yeast cells have to be fixed and the cell wall removed. Annexin V staining for the apoptosis-associated exposition of phosphatidyl serine also requires removal of the cell wall. Because the sensitive spheroplasts are easily damaged, integrity must be tested in parallel by a propidium iodine exclusion assay.

The level of intracellular reactive oxygen species (ROS) accumulation can be monitored by the incubation of cells with dihydrorhodamine 123 or dihydroethidium and by counting under a microscope or quantification in an appropriate fluorescent reader. Notably, these dyes are not able to detect specific ROS, and their signal is probably affected by metabolic rates.

Chromatin condensation and margination can be observed microscopically after DAPI staining or by using an electron microscope. Furthermore, fluorescence activated cell sorting (FACS) analysis is routinely used for ROS, annexin V, and propidium iodine staining and for the appearance of a sub-G0/G1 population.