Asymmetry of PI(3,5)P ₂ is critical for asymmetry of vacuolar pH. (A) Assessing relative acidity of mother and daughter vacuoles during the cell cycle of wildtype cells (MHY283). v-SEP-to-mCherry ratios (v-SEP/mCherry) of daughter and mother vacuoles serve as an in vivo pH sensor. The graph shows the mean v-SEP/mCherry ratios of five cells. Error bars are standard errors of the mean. Cells are aligned with respect to the end of vacuole segregation (t = 0). Selected time points from the time series of a representative cell are shown. White arrow marks the time of vacuole segregation (t = 0). The periods in which PI(3,5)P₂ asymmetry increases (increases), reduces (red.), and disappears are indicated on the graph based on Fig. 1. Data come from two independent experiments. Scale bars: 3 μm. (B) Representative still images and box-and-whisker plots of v-SEP/mCherry ratios between mother and daughter vacuole in WT (MHY283), vac7∆ (MHY291), fab1-ha (MHY257), and vac7∆ fab1-ha (MHY296). Data shown are from two independent experiments. Boxes show 25th and 75th percentiles, whiskers indicate 10th and 90th percentiles, and the line in the boxes represents the median. Ordinary one-way ANOVA was performed with Fisher’s LSD test. ****P < 0.0001, **P = 0.0087, ns: non-significant with P ≥ 0.6617. Scale bar: 3 μm. BF: bright field, m: mother, d: daughter.