Figure S2.
Meiotic autophagy is coupled to meiosis progression. (A and B) Shown are total mNG-Atg8 (A) and vacuolar mNG-Atg8 signal (line; B) with the standard deviations (shade) aligned to first Cdc14 release (anaphase I, marked by red dashed line). Cells from experiments in Fig. 1 C were divided into three groups based on how fast they went through meiosis (pink, 10% of the total, the fastest; gray, 10% of the total, the slowest; blue, 80% of the total, the middle; total, n = 303 cells; Kolmogorov–Smirnov test, P = 0.05). (C) Schematic of the chemical-genetic strategy for inhibiting the kinase activity of Atg1-as. Atg1 was genetically mutated to its Shokat allele, Atg1-M102G, with the bulky gatekeeper residue in the ATP-binding pocket of Atg1 replaced to create a functional ATP-analog–sensitive allele of Atg1 (Atg1-as). 1NM-PP1, a membrane-permeable ATP analog, can specifically inhibit Atg1-as kinase activity in a live cell. (D) Graph of Gal-NDT80–synchronized meiosis showing the percentage of cells at metaphase I (M-I), anaphase I (A-I), metaphase II (M-II), and anaphase II (A-II) at indicated time points. Cells derived every 0.5 h after NDT80 induction (t = 0 h) were fixed and subjected to IF staining (IF: α-tubulin/DAPI) as described in Materials and methods. (E) IB of meiotic cell lysates with indicated antibodies, showing the effect of induced Atg13-8SA (OE) on mCherry-GFP cleavage at indicated time relative to NDT80 induction. Left: Schematic of simultaneously induced expression of Atg13-8SA, mCherry-GFP, and NDT80 by 1 µM β-estradiol at 12 h in SPM (t = 0 h, induction). Atg13-8SA stimulates mCherry-GFP cleavage. (F) IF of Tub1 showing percentage of control (dashed lines) and Atg13-8SA (solid lines) cells reaching anaphase I (A-I, blue) and anaphase II (A-II, yellow) at indicated time points. Arrows mark A-I peaks; Atg13-8SA shortens the time to reach A-I peak; pattern of tubulin was quantified every 0.5 h after NDT80 induction (t = 0 h; n ≥ 100 cells at each time point). (G) Whole cell extracts derived from Gal-NDT80–synchronized meiotic cells were analyzed by IB with α-FLAG and α-Hxk1 antibodies. Graph at the bottom shows the quantification of FLAG-Atg1-as IB intensity with value (blue) in arbitrary units normalized by Hxk1 IB intensity. FLAG-Atg1-as level during meiotic divisions is relatively stable. (H) Atg1-as cells during synchronized meiosis (+ β-estradiol) or prophase I arrest (– β-estradiol) were collected at indicated time points. After Atg1 kinase assay, as diagrammed in Fig. 3 A, the whole-cell lysates were subjected to IB with indicated antibodies. NC, no supplemented ATP. (I and J) Percentage of cells with mNG-Atg8 puncta under vegetative growth (SD; I), nitrogen starvation (SD-N; J) or prophase I arrest (12 h in SPM as t = 0 h; I). n ≥ 300 cells at each time point from three replicate experiments; t test; ***, P ≤ 0.001. A.U., arbitrary unit. Source data are available for this figure: SourceData FS2.

Meiotic autophagy is coupled to meiosis progression. (A and B) Shown are total mNG-Atg8 (A) and vacuolar mNG-Atg8 signal (line; B) with the standard deviations (shade) aligned to first Cdc14 release (anaphase I, marked by red dashed line). Cells from experiments in Fig. 1 C were divided into three groups based on how fast they went through meiosis (pink, 10% of the total, the fastest; gray, 10% of the total, the slowest; blue, 80% of the total, the middle; total, n = 303 cells; Kolmogorov–Smirnov test, P = 0.05). (C) Schematic of the chemical-genetic strategy for inhibiting the kinase activity of Atg1-as. Atg1 was genetically mutated to its Shokat allele, Atg1-M102G, with the bulky gatekeeper residue in the ATP-binding pocket of Atg1 replaced to create a functional ATP-analog–sensitive allele of Atg1 (Atg1-as). 1NM-PP1, a membrane-permeable ATP analog, can specifically inhibit Atg1-as kinase activity in a live cell. (D) Graph of Gal-NDT80–synchronized meiosis showing the percentage of cells at metaphase I (M-I), anaphase I (A-I), metaphase II (M-II), and anaphase II (A-II) at indicated time points. Cells derived every 0.5 h after NDT80 induction (t = 0 h) were fixed and subjected to IF staining (IF: α-tubulin/DAPI) as described in Materials and methods. (E) IB of meiotic cell lysates with indicated antibodies, showing the effect of induced Atg13-8SA (OE) on mCherry-GFP cleavage at indicated time relative to NDT80 induction. Left: Schematic of simultaneously induced expression of Atg13-8SA, mCherry-GFP, and NDT80 by 1 µM β-estradiol at 12 h in SPM (t = 0 h, induction). Atg13-8SA stimulates mCherry-GFP cleavage. (F) IF of Tub1 showing percentage of control (dashed lines) and Atg13-8SA (solid lines) cells reaching anaphase I (A-I, blue) and anaphase II (A-II, yellow) at indicated time points. Arrows mark A-I peaks; Atg13-8SA shortens the time to reach A-I peak; pattern of tubulin was quantified every 0.5 h after NDT80 induction (t = 0 h; n ≥ 100 cells at each time point). (G) Whole cell extracts derived from Gal-NDT80–synchronized meiotic cells were analyzed by IB with α-FLAG and α-Hxk1 antibodies. Graph at the bottom shows the quantification of FLAG-Atg1-as IB intensity with value (blue) in arbitrary units normalized by Hxk1 IB intensity. FLAG-Atg1-as level during meiotic divisions is relatively stable. (H) Atg1-as cells during synchronized meiosis (+ β-estradiol) or prophase I arrest (– β-estradiol) were collected at indicated time points. After Atg1 kinase assay, as diagrammed in Fig. 3 A, the whole-cell lysates were subjected to IB with indicated antibodies. NC, no supplemented ATP. (I and J) Percentage of cells with mNG-Atg8 puncta under vegetative growth (SD; I), nitrogen starvation (SD-N; J) or prophase I arrest (12 h in SPM as t = 0 h; I). n ≥ 300 cells at each time point from three replicate experiments; t test; ***, P ≤ 0.001. A.U., arbitrary unit. Source data are available for this figure: SourceData FS2.

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