Alena Pichova

Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT‐mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro‐apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild‐type yeast mother cells.

The influence of oxygen toxicity on yeast mother cell-specific aging

The effect of deleting both catalase genes and of increased oxygen as well as paraquat (a pro-oxidant) on the replicative life span of yeast mother cells has been investigated to test the so-called oxygen theory of aging. This is well established in higher organisms, but has not been extensively tested in the unicellular yeast model system. Life span determinations were performed in ambient air or in a controlled atmosphere (55% oxygen) and an isogenic series of strains deleted for one or both yeast catalases was used and compared with wild type. In the absence of cellular catalase, increased oxygen caused a marked decrease in life span that could be completely reversed by adding 1 mM GSH, a physiological antioxidant, to the yeast growth medium. In a second unrelated strain, the effects were similar although even the wild type showed a decrease in life span when oxygen was increased. The effect could again be compensated by addition of extracellular GSH. Our results show that manipulating the detoxification of reactive oxygen species has a profound effect on yeast aging. These findings are discussed in the light of recent results relating to oxygen toxicity in the aging process of higher organisms.

Stratification of yeast cells during chronological aging by size points to the role of trehalose in cell vitality.

Cells of the budding yeast Saccharomyces cerevisiae undergo a process akin to differentiation during prolonged culture without medium replenishment. Various methods have been used to separate and determine the potential role and fate of the different cell species. We have stratified chronologically-aged yeast cultures into cells of different sizes, using centrifugal elutriation, and characterized these subpopulations physiologically. We distinguish two extreme cell types, very small (XS) and very large (L) cells. L cells display higher viability based on two separate criteria. They respire much more actively, but produce lower levels of reactive oxygen species (ROS). L cells are capable of dividing, albeit slowly, giving rise to XS cells which do not divide. L cells are more resistant to osmotic stress and they have higher trehalose content, a storage carbohydrate often connected to stress resistance. Depletion of trehalose by deletion of TPS2 does not affect the vital characteristics of L cells, but it improves some of these characteristics in XS cells. Therefore, we propose that the response of L and XS cells to the trehalose produced in the former differs in a way that lowers the vitality of the latter. We compare our XS- and L-fraction cell characteristics with those of cells isolated from stationary cultures by others based on density. This comparison suggests that the cells have some similarities but also differences that may prove useful in addressing whether it is the segregation or the response to trehalose that may play the predominant role in cell division from stationary culture.

Aged Yeast Mother Cells Show Markers of Apoptosis

METHODS. We have developed a new method for bulk isolation of old and young yeast cells by elutriation. The method is based on cell size only but yields remarkably pure old and young cell fractions. To characterize these cells, we used standard methods of fluorescence microscopy staining for ROS with dihydrorhodamine123 (DHR). The TUNEL methods as well as staining with annexinV were used to detect markers of apoptosis. Fluorescent staining with Calcofluor White was used to reveal bud scars. Life spans were determined by micromanipulation as described (2). RESULTS. The fraction containing large cells had a median lifespan of about 3.5 generations. About 10-20% of these cells were in their last cell cycle. Cells with more than 20 bud scars could be seen. The cytoskeleton was frequently abnormal pointing to loss of cell polarity. The fraction containing small cells in the same strain displayed a median lifespan of about 25 generations identical to the standard lifespan of the strain. The small cells showed just one birth scar and were normal in nuclear morphology.