Figure 6. Pkc1 prevents cell lysis by promoting membrane dissociation of Ste5 and Far1 upon cell–cell fusion. (A) The relative mating efficiency of ste5Δ cells expressing either WT Ste5 or Ste5S185A was measured in media where only diploid cells can grow. The SD was determined from at least three experiments. (B) Schematic of the microfluidic device used to observe the mating process. Cells were captured in pockets (magnified inset) and imaged by live-cell microscopy. The pillar array (1,008 traps/array) allows one to trap cells and visualize mating. (C and D) Zygotes resulting from the indicated crosses were followed after fusion and scored for cell lysis by microscopy. (C) The percentage of viable diploids is plotted as a function of time after cell–cell fusion; ≥100 zygotes from three different experiments were analyzed for each cross. The error bars indicate SD of three independent experiments, and significance was determined for the 150-min time point by a t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) Zygotes were assessed for emergence of their first bud after fusion event. Box and whisker plots show median and first and third quartiles, with the outlier 5th and 95th percentiles indicated as symbols (filled circles). Significance was determined by ANOVA and a t test (*, P ≤ 0.05; **, P ≤ 0.01). (E) 3xGFP-tagged Ste5 expressed in MATa cells was localized by fluorescence microscopy during cell–cell fusion with unlabeled MATα-partners. Images were taken at the indicated times with t = 0 defined when cell–cell contact is detected. Successful cell–cell fusion was monitored by the appearance of GFP-tagged proteins in the unlabeled mating partner. (F and G) The levels of 3xGFP-tagged WT Ste5 and Ste5S185A at the site of cell–cell fusion were quantified, and relative intensity with SD compared with the cytoplasmic signal as a function of time was plotted (F). t = 0 was defined as cell–cell fusion, monitored by the appearance of GFP-tagged proteins in the unlabeled mating partner. Zygotes were assessed for the time of localized GFP residence. The error bars indicate SD of three independent experiments, and significance was determined by a t test at the indicated time points (*, P ≤ 0.05). (G) Box and whisker plots show median and first and third quartiles, with the outlier 5th and 95th percentiles indicated as symbols (filled circles); ≥70 cells were analyzed, and a t test was used to determine significance (*, P ≤ 0.05).
Figure 6.

Pkc1 prevents cell lysis by promoting membrane dissociation of Ste5 and Far1 upon cell–cell fusion. (A) The relative mating efficiency of ste5Δ cells expressing either WT Ste5 or Ste5S185A was measured in media where only diploid cells can grow. The SD was determined from at least three experiments. (B) Schematic of the microfluidic device used to observe the mating process. Cells were captured in pockets (magnified inset) and imaged by live-cell microscopy. The pillar array (1,008 traps/array) allows one to trap cells and visualize mating. (C and D) Zygotes resulting from the indicated crosses were followed after fusion and scored for cell lysis by microscopy. (C) The percentage of viable diploids is plotted as a function of time after cell–cell fusion; ≥100 zygotes from three different experiments were analyzed for each cross. The error bars indicate SD of three independent experiments, and significance was determined for the 150-min time point by a t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (D) Zygotes were assessed for emergence of their first bud after fusion event. Box and whisker plots show median and first and third quartiles, with the outlier 5th and 95th percentiles indicated as symbols (filled circles). Significance was determined by ANOVA and a t test (*, P ≤ 0.05; **, P ≤ 0.01). (E) 3xGFP-tagged Ste5 expressed in MATa cells was localized by fluorescence microscopy during cell–cell fusion with unlabeled MATα-partners. Images were taken at the indicated times with t = 0 defined when cell–cell contact is detected. Successful cell–cell fusion was monitored by the appearance of GFP-tagged proteins in the unlabeled mating partner. (F and G) The levels of 3xGFP-tagged WT Ste5 and Ste5S185A at the site of cell–cell fusion were quantified, and relative intensity with SD compared with the cytoplasmic signal as a function of time was plotted (F). t = 0 was defined as cell–cell fusion, monitored by the appearance of GFP-tagged proteins in the unlabeled mating partner. Zygotes were assessed for the time of localized GFP residence. The error bars indicate SD of three independent experiments, and significance was determined by a t test at the indicated time points (*, P ≤ 0.05). (G) Box and whisker plots show median and first and third quartiles, with the outlier 5th and 95th percentiles indicated as symbols (filled circles); ≥70 cells were analyzed, and a t test was used to determine significance (*, P ≤ 0.05).

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