Pkc1 protects cells from lysis upon mechanostress during pheromone exposure through inhibition of the mating pathway. (A) Haploid mating-type a WT cells were exposed to α-factor for 100 min, followed by 15-min pretreatment with DMSO or 7.5 µM of the Pkc1 inhibitor cercosporamide (cerc.). Then, mechanostress or no stress was applied for 30 min. Lysis of shmooing cells was visualized by phase-contrast microscopy and staining with Trypan blue dye. (B) Quantification of cell lysis in shmooing WT cells, treated as in A; >150 cells in at least three independent experiments were quantified for each condition and shown as percentage of lysed cells. Error bars indicate SEM, and significance was determined by t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (C) Quantification of cell lysis in shmooing WT cells harboring or not (control) a plasmid expressing dominant-negative Pkc1 (Pkc1K853R) from the inducible GAL1 promoter. Cells growing in log phase in 2% raffinose were induced with 2% galactose for 2 h followed by exposure to α-factor for 100 min. Mechanostress was applied and cell lysis analyzed as described in B. (D) WT cells expressing the Fus3 SKAR and Hta2-CFP reporters were treated with pheromone and 7.5 µM cercosporamide (cerc.) as in A. Nuclear accumulation of the Fus3 SKAR reporting Fus3 activity after 30 min of mechanostress was monitored microscopically and quantified in ≥150 cells in three independent experiments. Error bars indicate SEM, and significance was determined by a t test (*, P ≤ 0.05). Representative images before and after mechanostress are shown below. (E) WT cells expressing the Fus3 SKARS and Hta2-CFP reporters were treated with pheromone and subsequently with 7.5 µM cercosporamide (cerc.) as described in A, and Fus3 activity was quantified in lysing and protected cells. Cells failing to relocalize the reporter to the nucleus under mechanostress were scored as high Fus3 activity; ≥150 cells were analyzed for each condition in three independent experiments and are shown as percentage of high or low Fus3 activity. Error bars indicate SEM. Note that cells unable to down-regulate Fus3 activity are prone to cell lysis. (F) WT cells expressing the chemically inhibitable Fus3-as allele from the endogenous locus were treated with α-factor for 100 min followed by pretreatment with 7.5 µM cercosporamide (cerc.) and 5 µM NaPP1. Then mechanostress was applied. Lysis of ≥150 pheromone-responsive cells was scored for each condition in three independent experiments and is shown as percentage of lysed cells. Error bars indicate SEM, and significance was determined by a t test (***, P ≤ 0.001). (G) Quantification of cell lysis upon mechanostress in WT or mpk1Δ cells treated as in B. At least 150 cells in at least three independent experiments were analyzed for each condition and are shown as percentage of lysed cells. Error bars indicate SEM, and significance was verified by a t test (**, P ≤ 0.01; ***, P ≤ 0.001). Genetic background and growth conditions in this experiment are the same as in B, and thus, the data for the WT conditions were combined. (H) Cells expressing Ste5-tV under its endogenous promotor in WT or mpk1Δ cells were treated as in A, and Ste5-tV was visualized microscopically in microfluidic chips during 30 min of mechanostress. Loss of Ste5-tV at shmoo tips was quantified in ≥150 cells for each condition in at least three independent experiments, and shown as percentage of cells with dispersed Ste5 localization. Error bars indicate SEM and significance was validated by a t test (**, P ≤ 0.01; ***, P ≤ 0.001). (I) Pkc1-dependent signaling protects mating cells against mechanostress. Pkc1 triggers loss of Ste5 from shmoo tips by an Mpk1-independent mechanism, leading to reduced Fus3 activity.
