Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts

Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. In this study, using budding yeast and normal human fibroblasts, we found that cellular senescence—stable cell cycle arrest contributing to organismal aging—is the long-term outcome of PMD. Our genetic screening using budding yeast unexpectedly identified a close genetic association between PMD response and replicative lifespan regulations. Furthermore, PMD limits replicative lifespan in budding yeast; upregulation of membrane repair factors ESCRT-III (SNF7) and AAA-ATPase (VPS4) extends it. In normal human fibroblasts, PMD induces premature senescence via the Ca2+–p53 axis but not the major senescence pathway, DNA damage response pathway. Transient upregulation of ESCRT-III (CHMP4B) suppressed PMD-dependent senescence. Together with mRNA sequencing results, our study highlights an underappreciated but ubiquitous senescent cell subtype: PMD-dependent senescent cells.

V-ATPase produces a proton gradient across the vacuolar membrane, enabling Ca 2+ uptake into the vacuole; the mutants lucking functional V-ATPase show high cytoplasmic Ca 2+ levels 65 .Since Ca 2+ influx at the damage site is essential for membrane resealing in higher eukaryotes 6,7 , SDS sensitivity in V-ATPase mutants may be explained by the high cytosolic Ca 2+ concentration in V-ATPase mutants, preventing membrane resealing.Indeed, in our DAPI penetration assay (30 min incubation in YPD+SDS and quick wash with YPD, followed by 5 min incubation with DAPI), V-ATPase mutants (vma1∆ and vma13∆) showed high vma13∆: 63.0±8.6;Extended Data Fig. 4e) analogous to SDS and EGTA-treated wild type (Fig. 1a and b).These results raise a possibility that Ca 2+ influx-dependent membrane resealing is impaired in V-ATPase mutants.
To test the possibility that Ca 2+ homeostasis is defective in V-ATPase mutants, we monitored subcellular localization of Crz1-GFP, a Ca 2+ -responsive transcription factor that enters nucleus after various stimuli 66 .After the laser-induced cell wall and plasma membrane damage, Crz1-GFP entered the nucleus within 30 sec (Extended Data Fig. 4f and   g).In a V-ATPase mutant vma1∆, even before the laser damage Crz1-GFP signals at the nucleus was comparable to the peak levels of control cells and did not increase after the laser damage.These results support our interpretation that Ca 2+ -influx detection is impaired in vma1∆.
Since cho1∆, defective in phospholipid phosphatidylserine (PS) synthesis, was a screening hit, we examined whether PS is involved in plasma membrane/cell wall repair processes after the laser damage.Wild type yeast cells harboring a plasmid of Lact-C2-GFP, a PS marker, were subjected to the laser damage assay.We found that Lact-C2-GFP signal gradually accumulated at the damage site and peaked after ~13 min (Extended Data Fig. 4h and i), consistent with the idea that PS is involved in the plasma membrane/cell wall repair processes.
We next focused on pep3∆ and vps34∆.These mutants shared three common phenotypes: 1) the cell viability did not decrease after 2hr SDS treatment (Extended Data Fig. 4d), 2) the cells did not show DAPI penetration after 30 min SDS treatment (Extended Data Fig. 4e), and 3) in the laser damage experiment, a major repair protein Pkc1-GFP failed to be retained at the laser damage site (Extended Data Fig. 4j and k).These results suggest that Pep3 and Vps34 are required for the retention of Pkc1 but not for plasma membrane resealing immediately after the damage or initial Pkc1 recruitment to the damage site.
In summary, here we revealed four cellular processes during plasma membrane/cell wall damage response in budding yeast: 1) V-ATPase-dependent prevention of immediate cell death, 2) Crz1 nuclear import, 3) PS recruitment to the damage site, and 4) Pep3 and Vps34-dependent retention of Pkc1 (Supplementary Fig. 2).concentration that does not induce cell death after 24 hours of SDS treatment.This experiment was independently repeated three times with similar results.