Monitoring yeast regulated cell death: trespassing the point of no return to loss of plasma membrane integrity.

Acetic acid and hydrogen peroxide are the most common stimuli to induce apoptosis in yeast. The initial phase of this cell death process is characterized by the maintenance of plasma membrane integrity in cells that had already lost their viability. As loss of plasma membrane integrity is typically assessed by staining with propidium iodide (PI) after exposure of cells to a stimulus and cell viability is determined 48 h after plating, the percentage of cells with compromised plasma membrane integrity and c.f.u. counts often do not correlate. Herein, we developed a simple method to explore at what point after an apoptotic stimulus and plating cells do non-viable cells die as result of plasma membrane disruption, i.e., when cells surpass the point-of-no-return and undergo a secondary necrosis. The method consisted in washing cells and re-suspending them in stimulus-free medium after acetic acid and hydrogen peroxide treatments, to mimic transfer to plating, and then assessing plasma membrane integrity through PI staining. We show that, after the stimuli are removed, cells that had lost proliferative capacity but still maintained plasma membrane integrity continue the cell death process and later lose plasma membrane integrity when progressing to secondary necrosis. After exposure to hydrogen peroxide, cells undergo secondary necrosis preceded by Nhp6Ap-GFP cytosolic localization, in contrast to acetic acid exposure, where Nhp6Ap-GFP cytosolic localization mainly occurs simultaneously with an earlier emergence of secondary necrosis. In conclusion, the developed method allows monitoring the irreversible loss of plasma membrane integrity of dying apoptotic cells after the point-of-no-return is trespassed, and better characterize the process of secondary necrosis after apoptosis.