Figure 3.
Figure 3. Ndt80 and Ime2 regulate mitochondrial detachment. (A) Maximum-intensity projections of fixed pGAL-NDT80 GAL4.ER cells (UB9496) with NDT80 induced by addition of 1 µM β-estradiol (+Ndt80) or ethanol vehicle control (−Ndt80). Sporulation cultures were arrested for 5 h, then split and subjected to the indicated treatment. Cells were imaged 0 and 3 h after treatment. Mitochondria, Cit1-GFP; nuclei, Htb1-mCherry. Dashed lines: cell boundaries. (B) Maximum-intensity projections of fixed pGAL-NDT80 GAL4.ER cells containing wild-type IME2 (UB9158) or ime2-as1 (UB9844). Sporulation cultures were arrested for 5 h, then 1 µM β-estradiol and 20 µM 1-NA-PP1 were added simultaneously. Cells were imaged 0 and 3 h after treatment. Dashed lines: cell boundaries. (C and D) Execution point time course, with the timing of Ime2 inhibition varied relative to constant induction of pGAL-NDT80. (C) Top: Schematic of the execution point time course. Wild-type pGAL-NDT80 GAL4.ER (UB9158) and pGAL-NDT80 GAL4.ER ime2-as1 (UB9844) were induced to sporulate and arrested for 5 h, then treated with 1 µM β-estradiol to induce NDT80 (time = 0 h). Next, the ime2-as1 culture was split and treated with 20 µM 1-NA-PP1 at the indicated time points relative to NDT80 induction. DMSO served as the vehicle control and was added simultaneously with β-estradiol. The wild-type control was treated with 1-NA-PP1 and β-estradiol simultaneously. Bottom: Heat map showing the frequency of mitochondrial detachment at 0, 3, and 5 h after NDT80 induction (i.e., the time of fixation) for each of the execution points (n = 2,200 total cells counted, with n ≥ 32 per panel in the heat map). (D) Maximum-intensity projections from wild-type control and the 2.5 h execution point. Dashed lines: cell boundaries for meiosis II cells only. (E) Maximum-intensity projections of prophase I arrested cells (ndt80Δ, 5 h in SPO) containing wild-type IME2 (UB17328) or IME2-st (UB17330). Dashed lines: cell boundaries. (F) Quantification of mitochondrial morphology (percentage of cells with detached mitochondria) for the experiment in E (n = 100 per genotype). P < 0.0001, by Fisher’s exact test. Scale bars, 2 µm.

Ndt80 and Ime2 regulate mitochondrial detachment. (A) Maximum-intensity projections of fixed pGAL-NDT80 GAL4.ER cells (UB9496) with NDT80 induced by addition of 1 µM β-estradiol (+Ndt80) or ethanol vehicle control (−Ndt80). Sporulation cultures were arrested for 5 h, then split and subjected to the indicated treatment. Cells were imaged 0 and 3 h after treatment. Mitochondria, Cit1-GFP; nuclei, Htb1-mCherry. Dashed lines: cell boundaries. (B) Maximum-intensity projections of fixed pGAL-NDT80 GAL4.ER cells containing wild-type IME2 (UB9158) or ime2-as1 (UB9844). Sporulation cultures were arrested for 5 h, then 1 µM β-estradiol and 20 µM 1-NA-PP1 were added simultaneously. Cells were imaged 0 and 3 h after treatment. Dashed lines: cell boundaries. (C and D) Execution point time course, with the timing of Ime2 inhibition varied relative to constant induction of pGAL-NDT80. (C) Top: Schematic of the execution point time course. Wild-type pGAL-NDT80 GAL4.ER (UB9158) and pGAL-NDT80 GAL4.ER ime2-as1 (UB9844) were induced to sporulate and arrested for 5 h, then treated with 1 µM β-estradiol to induce NDT80 (time = 0 h). Next, the ime2-as1 culture was split and treated with 20 µM 1-NA-PP1 at the indicated time points relative to NDT80 induction. DMSO served as the vehicle control and was added simultaneously with β-estradiol. The wild-type control was treated with 1-NA-PP1 and β-estradiol simultaneously. Bottom: Heat map showing the frequency of mitochondrial detachment at 0, 3, and 5 h after NDT80 induction (i.e., the time of fixation) for each of the execution points (n = 2,200 total cells counted, with n ≥ 32 per panel in the heat map). (D) Maximum-intensity projections from wild-type control and the 2.5 h execution point. Dashed lines: cell boundaries for meiosis II cells only. (E) Maximum-intensity projections of prophase I arrested cells (ndt80Δ, 5 h in SPO) containing wild-type IME2 (UB17328) or IME2-st (UB17330). Dashed lines: cell boundaries. (F) Quantification of mitochondrial morphology (percentage of cells with detached mitochondria) for the experiment in E (n = 100 per genotype). P < 0.0001, by Fisher’s exact test. Scale bars, 2 µm.

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