Purification of Yeast Spores to Investigate Their Dynamics of Activation

Abstract

Germination is an important developmental process that supports resumption of growth in dormant spores. The study of the mechanisms underlying germination requires a pure spore population devoid of other cell types. This article describes the sporulation of wild Saccharomyces cerevisiae and Saccharomyces paradoxus strains, and the isolation and purification of ascospores. We also describe a method to synchronously induce germination in a spore population as well as to measure spore activation. This procedure can be applied, for example, to the study of environmental conditions that trigger germination.

Breaking spore dormancy in budding yeast transforms the cytoplasm and the solubility of the proteome

“…The hallmark of the completion of germination and the return to vegetative growth is bud emergence, which occurs at about 6 h after induction of germination ( Fig 1A ). Spores’ transition from high to low refractility correlates with the decrease in optical density at 595 nm (A 595 ) of the pure spore culture [ 30 ], with the minimal values reached about 3 h after induction ( Fig 1B ). Then, subsequent growth leads to an increase of optical density.…”

“…Sporulation was conducted on sporulation medium plates containing 1% potassium acetate, 0.1% yeast extract, 0.01% glucose, and 2% agar and spores were further purified on Percoll gradient (Sigma) as previously described [ 30 ]. Germination was induced by transferring spores to YPD.…”

Breaking dormancy in spores of budding yeast transforms its cytoplasm and the solubility of its proteome

“…The hallmark of the completion of germination and the return to vegetative growth is bud emergence, which occurs at about 6 hours after induction of germination (Figure 1A). Cells’ transition from high to low refractility correlates with the decrease in optical density at 595 nm ( A 595 ) of the pure spore culture (Plante and Landry, 2020b), with the minimal values reached about 3 hours after induction (Figure 1B). One of the adaptive features of spores is their resistance to heat.…”

“…Sporulation was conducted on sporulation medium plates containing 1% potassium acetate, 0.1% yeast extract, 0.01% glucose, and 2% agar and spores were further purified on Percoll gradient (Sigma) as previously described (Plante and Landry, 2020b). Germination was induced by transferring spores to YPD.…”

“…Resistance measurements in spores during germination (Figure 1B) were conducted as described previously (Plante and Landry, 2020b). Briefly, freshly purified wild spores were induced in germination in YPD medium, and at the indicated time following induction cells were sampled.…”