Figure 1.
PI(3,5)P2sensor SnxA indicates daughter vacuole-specific PI(3,5)P2synthesis during late mitosis. (A–C) Analysis of SnxA-GFP colocalization with the vacuolar marker Vph1-3xmCherry using still images of log-phase cell populations. Box-and-whisker plots of PCC and representative still images are presented. PCC values were obtained using the EzColocalization plugin in ImageJ by selecting the vacuoles based on Vph1-3xmCherry as a vacuole marker (see Materials and methods for details). PCC provides a metric for the likelihood of co-existence of SnxA-GFP and Vph1-3xmCherry signals in the given area, thus objectively assessing colocalization. Data shown are a comparison of wildtype cells (WT) and PI(3,5)P2-deficient mutants (MHY286, MHY304, MHY302, and AAY007) in A, comparison of these cells (−) with cells expressing fab1-ha (MHY286, MHY288, MHY304, MHY305, MHY302, and MHY303) in B, and comparison of basal conditions (−) with hyperosmotic shock conditions (+) in C. Experiments shown in A–C were performed together, thus the controls (WT, vac7∆, vac14∆, and fab1∆) are identical in all graphs. In box-and-whisker plots, boxes extend from the 25th to 75th percentiles. Lines inside the boxes are the median. Whiskers point to the 10th and 90th percentiles. Ordinary one-way ANOVA was performed with Fisher’s LSD test. ****P < 0.0001, ns: non-significant with P ≥ 0.1. n: sample size. Data shown come from four independent experiments. (D and E) Analysis of the effect of hyperosmotic shock on SnxA-GFP vacuole periphery localization through time-lapse microscopy. Mean PCC values of SnxA-GFP and Vph1-3xmCherry during the time-lapse series are plotted (E) along with images from selected time points of representative cells (D). The graph represents the mean of PCC values obtained from 20 cells (MHY286) subjected to 0.9 M NaCl treatment. Error bars show the standard error of the mean. Red arrows indicate the time point of NaCl addition to the culture dish. Scale bars: 3 μm, BF: bright field. (F) Box-and-whisker plots showing PCC values of SnxA-GFP colocalization with the vacuolar marker Vph1-3xmCherry on the mother vacuole (MV) and daughter vacuole (DV) in log-phase WT cells (MHY286) with segregated vacuoles. Boxes show the 25th to 75th percentiles, whiskers point to the 10th and 90th percentiles, and lines inside the boxes are the median. See Fig. 1 A for representative images. An unpaired two-tailed t test was performed. ****P < 0.0001. Data shown come from three independent experiments. (G and H) Time-lapse microscopy analysis of the SnxA-GFP localization at the vacuole periphery during the cell cycle of WT cells (MHY286). Image series of selected time points from a representative cell is shown in B. Time-dependent changes in PCC values of SnxA-GFP colocalization with the vacuolar marker Vph1-3xmCherry on the mother and daughter vacuoles are shown in the graph (H). The graph represents the mean of individual cells aligned with respect to the end of vacuole segregation (t = 0). Error bars are the standard error of the mean. The white arrow in G indicates the completion 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. Data come from two independent experiments. (I and J) Analysis of SnxA-GFP localization on the mother and daughter vacuole periphery with respect to the timing of SnxA-GFP signal appearance on the daughter vacuole (DV) relative to the end of vacuole segregation (t = 0) (I), and the duration of SnxA-GFP localization on the mother vacuole (MV) and daughter vacuole (DV) periphery (J). Circles on scatter plots indicate measurements of individual cells. Black lines in scatter blots are mean and standard deviation. Data come from two independent experiments. (K) Time-lapse microscopy-based analysis of SnxA-mCherry localization with respect to spindle elongation in WT cells containing GFP-Tub1 (MHY295). Selected time points from a representative cell are shown. White arrow marks the time point of anaphase onset (t = 0) designated by the start of fast spindle elongation. Yellow arrow marks the timing of spindle breakdown as an indicative of mitotic exit. The graph shows the timing of SnxA-mCherry accumulation on the daughter vacuole (arrival at DV) with respect to the anaphase onset (t = 0), and SnxA-mCherry disappearance from the daughter vacuole (departure from DV) with respect to the spindle breakdown (t = 20). Scale bars: 3 μm. n: sample size. m: mother, d: daughter.

PI(3,5)P 2 sensor SnxA indicates daughter vacuole-specific PI(3,5)P 2 synthesis during late mitosis. (A–C) Analysis of SnxA-GFP colocalization with the vacuolar marker Vph1-3xmCherry using still images of log-phase cell populations. Box-and-whisker plots of PCC and representative still images are presented. PCC values were obtained using the EzColocalization plugin in ImageJ by selecting the vacuoles based on Vph1-3xmCherry as a vacuole marker (see Materials and methods for details). PCC provides a metric for the likelihood of co-existence of SnxA-GFP and Vph1-3xmCherry signals in the given area, thus objectively assessing colocalization. Data shown are a comparison of wildtype cells (WT) and PI(3,5)P2-deficient mutants (MHY286, MHY304, MHY302, and AAY007) in A, comparison of these cells (−) with cells expressing fab1-ha (MHY286, MHY288, MHY304, MHY305, MHY302, and MHY303) in B, and comparison of basal conditions (−) with hyperosmotic shock conditions (+) in C. Experiments shown in A–C were performed together, thus the controls (WT, vac7∆, vac14∆, and fab1∆) are identical in all graphs. In box-and-whisker plots, boxes extend from the 25th to 75th percentiles. Lines inside the boxes are the median. Whiskers point to the 10th and 90th percentiles. Ordinary one-way ANOVA was performed with Fisher’s LSD test. ****P < 0.0001, ns: non-significant with P ≥ 0.1. n: sample size. Data shown come from four independent experiments. (D and E) Analysis of the effect of hyperosmotic shock on SnxA-GFP vacuole periphery localization through time-lapse microscopy. Mean PCC values of SnxA-GFP and Vph1-3xmCherry during the time-lapse series are plotted (E) along with images from selected time points of representative cells (D). The graph represents the mean of PCC values obtained from 20 cells (MHY286) subjected to 0.9 M NaCl treatment. Error bars show the standard error of the mean. Red arrows indicate the time point of NaCl addition to the culture dish. Scale bars: 3 μm, BF: bright field. (F) Box-and-whisker plots showing PCC values of SnxA-GFP colocalization with the vacuolar marker Vph1-3xmCherry on the mother vacuole (MV) and daughter vacuole (DV) in log-phase WT cells (MHY286) with segregated vacuoles. Boxes show the 25th to 75th percentiles, whiskers point to the 10th and 90th percentiles, and lines inside the boxes are the median. See Fig. 1 A for representative images. An unpaired two-tailed t test was performed. ****P < 0.0001. Data shown come from three independent experiments. (G and H) Time-lapse microscopy analysis of the SnxA-GFP localization at the vacuole periphery during the cell cycle of WT cells (MHY286). Image series of selected time points from a representative cell is shown in B. Time-dependent changes in PCC values of SnxA-GFP colocalization with the vacuolar marker Vph1-3xmCherry on the mother and daughter vacuoles are shown in the graph (H). The graph represents the mean of individual cells aligned with respect to the end of vacuole segregation (t = 0). Error bars are the standard error of the mean. The white arrow in G indicates the completion 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. Data come from two independent experiments. (I and J) Analysis of SnxA-GFP localization on the mother and daughter vacuole periphery with respect to the timing of SnxA-GFP signal appearance on the daughter vacuole (DV) relative to the end of vacuole segregation (t = 0) (I), and the duration of SnxA-GFP localization on the mother vacuole (MV) and daughter vacuole (DV) periphery (J). Circles on scatter plots indicate measurements of individual cells. Black lines in scatter blots are mean and standard deviation. Data come from two independent experiments. (K) Time-lapse microscopy-based analysis of SnxA-mCherry localization with respect to spindle elongation in WT cells containing GFP-Tub1 (MHY295). Selected time points from a representative cell are shown. White arrow marks the time point of anaphase onset (t = 0) designated by the start of fast spindle elongation. Yellow arrow marks the timing of spindle breakdown as an indicative of mitotic exit. The graph shows the timing of SnxA-mCherry accumulation on the daughter vacuole (arrival at DV) with respect to the anaphase onset (t = 0), and SnxA-mCherry disappearance from the daughter vacuole (departure from DV) with respect to the spindle breakdown (t = 20). Scale bars: 3 μm. n: sample size. m: mother, d: daughter.

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