Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae

Abstract

The variable stress-sensitivity of individual cells within pure cultures is widely noted but generally unexplained. Here, factors determining the heterogeneous susceptibility to copper toxicity in Saccharomyces cerevisiae were examined with a rapid nonperturbing approach based on flow cytometry. By determination of the DNA content (with propidium iodide) in cell fractions gated by forward angle light scatter (an indicator of the cell volume), it was shown that forward angle light scatter measurements gave an approximation of the cell cycle stage. Thus, our observation that cells in different forward angle light scatter fractions displayed differing Cu-sensitivities indicated that heterogeneous Cu-sensitivity is a function of the cell cycle stage. Furthermore, cells sorted by their Cu-sensitivity and -resistance and subsequently analyzed for DNA content were found predominantly to occupy G 1 /S and G 2 /M cell cycle stages, respectively. The oxidant-sensitive probe 2P,7P-dichlorodihydrofluorescein diacetate was used to show that the Cu-sensitivity of G 2 /M phase S. cerevisiae was correlated with greater levels of pre-existing reactive oxygen species in these cells. The results indicate that differential Cu-sensitivity in a S. cerevisiae culture is linked to the cell cycle stage and this link may be determined partly by cell cycle-dependent fluctuations in basal reactive oxygen species generation. ß

Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry

“…1a). Previous results on DNA distributions (Howlett & Avery, 1999 ;Fortuna et al, 2000) showed that these regions are related to the different phases of the cell cycle ; the A, B and C gated regions correspond to cells in phases G0\G1, S and G2\M, respectively. The fluorescence obtained in each gated region gives rise to three different distributions (Fig.…”

A Temperature-Sensitive dcw1 Mutant of Saccharomyces cerevisiae Is Cell Cycle Arrested with Small Buds Which Have Aberrant Cell Walls

“…Cells were grown in YPAD to a density of 5 ϫ 10 6 to 10 ϫ 10 6 cells/ml at 25°C and incubated at 37°C for 3 h. Culture samples corresponding to 10 7 cells were washed in 1 ml of 0.2 M Tris-HCl (pH 7.5), fixed in 1 ml of 0.2 M Tris-HCl (pH 7.5) containing 70% ethanol, washed in 1 ml of 0.2 M Tris-HCl (pH 7.5), dissolved in 1 ml of 0.2 M Tris-HCl (pH 7.5) containing 0.25 mg of RNase A (Wako)/ml, incubated at 50°C for 1 h, mixed with 20 l of 50-mg/ml proteinase K (Wako), incubated at 50°C for 1 h, pelleted by centrifugation at 10,000 ϫ g for 2 min, dissolved in 1 ml of 0.2 M Tris-HCl (pH 7.5) containing 16 g of propidium iodide/ml, incubated at room temperature for 30 min in a dark room, sonicated, and analyzed by flow cytometry (Coulter; Epics Elite ESP) (13).…”

Hydrocarbon Pollution: Effects on Living Organisms, Remediation of Contaminated Environments, and Effects of Heavy Metals Co-Contamination on Bioremediation

“…b. Effect of cell cycle - Howlett and Avery (1999) investigated the relationship between the cell volume (as the volume of the cells vary during the cell cycle) and the susceptibility to copper toxicity in Saccharomyces cerevisiae. The correlation between the cell volume (by examining the forward angle light scatter -FSC) and the cell cycle stage (by examining the DNA content) indicated that the largest (the cells about to undergo mitosis) were most resistant to Cu and the smallest (newly divided cells) cells were also relatively resistant to copper.…”