Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures

Abstract

Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G0 state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle.

Budding YeastSSD1-VRegulates Transcript Levels of Many Longevity Genes and Extends Chronological Life Span in Purified Quiescent Cells

“…However, the negative effects of auxotrophy that we observe do not appear to do be due to starvation for required compounds. Despite excess supplementation, the prototroph survives SP much longer than its parent auxotroph.Even with prototrophs, only a fraction of the cells in a SP culture attain the quiescent state (Allen et al, 2006). We also find that the yield of Q cells in these SP cultures diminishes with time in culture and varies with the genetic background.…”

“…Another variable that affects the life span of a SP culture is the fact that these cultures contain cells that are already dead and cells that are viable but cannot resume cell division (Herker et al, 2004;Allen et al, 2006). These cells obscure the properties of the quiescent cells in the population.…”

“…These cells obscure the properties of the quiescent cells in the population. To specifically assay the longevity of the Q cells, we have used a new purification procedure that separates Q cells from all others by virtue of their increased density (Allen et al, 2006). The cause of this increased density is not understood, but it serves as a convenient property by which to purify a population of cells that retain all the features associated with quiescence.…”

“…These Q cells arrest with G1 DNA content and maintain high levels of storage carbohydrates. They also show little or no indication of reactive oxygen species or apoptosis (Allen et al, 2006). By assaying the long-term survival of purified, prototrophic Q cells, we can obtain a direct measure of CLS and assess the influence of mutations on the longevity of quiescent cells.…”

“…Auxotrophy has a significant, but variable, negative impact on the reproductive capacity of both strains, and this deleterious effect is not mitigated by oversupplementation of required compounds. We conclude that in order to assess the effect of Ssd1 or any gene product on survival after nutrient exhaustion, the assays must be performed in prototrophs.Another variable that affects the life span of a SP culture is the fact that these cultures contain cells that are already dead and cells that are viable but cannot resume cell division (Herker et al, 2004;Allen et al, 2006). These cells obscure the properties of the quiescent cells in the population.…”