Figure 2.
Autophagy level influences meiosis kinetics and sporulation. (A) Heatmap analysis of total (top) and vacuolar (bottom) mNG-Atg8 levels of individual meiotic cells that completed meiosis, aligned to the onset of premeiotic DNA replication. (B) Left: Correlation plots of Atg8 (mNG-Atg8) level with the onset time of meiosis I (first Cdc14 release; top left) and meiosis II (second Cdc14 release; bottom left). Right: Correlation plots of Whi5 nuclear level with the onset time of meiosis I (top right) and meiosis II (bottom right). Cells transferred to SPM (0 h). (C) Schematic of simultaneously induced expression of Atg13-8SA (pZD: Atg13-8SA, for autophagy enhanced) and NDT80 (pGAL: NDT80, for synchronized metaphase I entry) by 1 µM β-estradiol. For GAL-NDT80−synchronized meiosis, 1 µM β-estradiol was added to meiotic cells after 12 h in SPM to synchronize the entry of meiotic divisions, depicted as t = 0 h of NDT80 induction. (D–G) Effects of Atg13-8SA on synchronized meiotic kinetics and sporulation. Control, strain without pZD: Atg13-8SA. (D and E) IB of Clb3-FLAG/Hxk1 in control and Atg13-8SA cells (D); IF of Tub1 showing percentage of control (dashed lines) and Atg13-8SA (solid lines) cells reaching metaphase I (M-I, green) and metaphase II (M-II, grey; E) at indicated time points. n ≥ 100. Arrows mark M-II peaks, Atg13-8SA shortens the time to reach M-II peak. (F) Percentage of tri-/tetranucleated cells (DAPI staining) after 6 h of NDT80 induction. n ≥ 300. Statistically significant differences: **, P ≤ 0.01; error bars represent standard deviations of three replicate experiments. (G) Sporulation efficiency (%) in control or Atg13-8SA cells after 48 h in SPM (n ≥ 300 cells; t test; ***, P ≤ 0.001). 1NM-PP1 treatment (5 µM, autophagy inhibition) at entry of metaphase I (NDT80 expression) abolished sporulation. A.U., arbitrary unit. Source data are available for this figure: SourceData F2.

Autophagy level influences meiosis kinetics and sporulation. (A) Heatmap analysis of total (top) and vacuolar (bottom) mNG-Atg8 levels of individual meiotic cells that completed meiosis, aligned to the onset of premeiotic DNA replication. (B) Left: Correlation plots of Atg8 (mNG-Atg8) level with the onset time of meiosis I (first Cdc14 release; top left) and meiosis II (second Cdc14 release; bottom left). Right: Correlation plots of Whi5 nuclear level with the onset time of meiosis I (top right) and meiosis II (bottom right). Cells transferred to SPM (0 h). (C) Schematic of simultaneously induced expression of Atg13-8SA (pZD: Atg13-8SA, for autophagy enhanced) and NDT80 (pGAL: NDT80, for synchronized metaphase I entry) by 1 µM β-estradiol. For GAL-NDT80−synchronized meiosis, 1 µM β-estradiol was added to meiotic cells after 12 h in SPM to synchronize the entry of meiotic divisions, depicted as t = 0 h of NDT80 induction. (D–G) Effects of Atg13-8SA on synchronized meiotic kinetics and sporulation. Control, strain without pZD: Atg13-8SA. (D and E) IB of Clb3-FLAG/Hxk1 in control and Atg13-8SA cells (D); IF of Tub1 showing percentage of control (dashed lines) and Atg13-8SA (solid lines) cells reaching metaphase I (M-I, green) and metaphase II (M-II, grey; E) at indicated time points. n ≥ 100. Arrows mark M-II peaks, Atg13-8SA shortens the time to reach M-II peak. (F) Percentage of tri-/tetranucleated cells (DAPI staining) after 6 h of NDT80 induction. n ≥ 300. Statistically significant differences: **, P ≤ 0.01; error bars represent standard deviations of three replicate experiments. (G) Sporulation efficiency (%) in control or Atg13-8SA cells after 48 h in SPM (n ≥ 300 cells; t test; ***, P ≤ 0.001). 1NM-PP1 treatment (5 µM, autophagy inhibition) at entry of metaphase I (NDT80 expression) abolished sporulation. A.U., arbitrary unit. Source data are available for this figure: SourceData F2.

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