Figure 8. Ime2 regulates MECA function in meiosis. (A and B) Immunoblots of MECA subunits in pGAL-NDT80 GAL4.ER. Strains were induced to sporulate and were released 5 h later from prophase I arrest by the addition of 1 µM β-estradiol to induce NDT80. Simultaneously, 20 µM 1-NA-PP1 was added to inhibit ime2-as1, with wild-type IME2 as the control. Band intensity quantifications normalized to the Hxk2 loading control and time course staging by DAPI staining for each time point are shown below the immunoblots. (A) Num1-3V5 immunoblot in wild type (UB12402) and ime2-as1 (UB12403). Num1 runs above the highest (190-kD) ladder band. (B) Mdm36-3V5 immunoblot in wild type (UB13851) and ime2-as1 (UB14546). (C) Maximum-intensity projections of live pGAL-NDT80 GAL4.ER cells containing wild-type IME2 (UB15124) or ime2-as1 (UB16047). Cells were induced to sporulate, then treated with 1 µM β-estradiol and 20 µM 1-NA-PP1 at 5 h. Images were acquired at the time of β-estradiol addition (0 h) or 3 h later. Dashed lines: cell boundaries. Mitochondria, mitoBFP. Nuclei, Htb1-mCherry. (D and E) Quantifications for the experiment in C. Analysis was restricted to cells that had entered the meiotic divisions (n = 20 cells per group). Medians are plotted as horizontal lines. The Mann–Whitney nonparametric test was used to test statistical significance. (D) Number of Num1-GFP foci per cell (see Materials and methods). (E) Whole-cell Num1-GFP fluorescence quantification from maximum-intensity projection, normalized to cell area. (F) Localization of Num1-mKate2 and GFP-Mdm36 in live pGAL-NDT80 GAL4.ER (UB18219) and pGAL-NDT80 GAL4.ER ime2-as1 (UB18221) cells. After 5 h in SPO medium, the cultures were treated with 1 µM β-estradiol and 20 µM 1-NA-PP1. Images were acquired 4 h later. Single planes in the z-axis are shown. Entry into the meiotic divisions was staged by the prospore membrane marker BFP-Spo2051–91. Note that prospore membranes are misshapen in the ime2 mutant. (G) Maximum-intensity projections of live wild-type IME2 ndt80Δ cells (UB16806) or IME2-st ndt80Δ cells (UB16808) arrested in prophase I for 5 h. Cells express the same cellular markers as in C. (H and I) Quantifications of the experiment in G (n = 20 cells per group). Medians are plotted as horizontal lines. The Mann–Whitney nonparametric test was used to test statistical significance. (H) Number of Num1-GFP foci per cell (see Materials and methods). (I) Whole-cell Num1-GFP fluorescence quantification from maximum-intensity projection, normalized to cell area. Scale bars, 2 µm; inset panels, 400 nm.
Figure 8.

Ime2 regulates MECA function in meiosis. (A and B) Immunoblots of MECA subunits in pGAL-NDT80 GAL4.ER. Strains were induced to sporulate and were released 5 h later from prophase I arrest by the addition of 1 µM β-estradiol to induce NDT80. Simultaneously, 20 µM 1-NA-PP1 was added to inhibit ime2-as1, with wild-type IME2 as the control. Band intensity quantifications normalized to the Hxk2 loading control and time course staging by DAPI staining for each time point are shown below the immunoblots. (A) Num1-3V5 immunoblot in wild type (UB12402) and ime2-as1 (UB12403). Num1 runs above the highest (190-kD) ladder band. (B) Mdm36-3V5 immunoblot in wild type (UB13851) and ime2-as1 (UB14546). (C) Maximum-intensity projections of live pGAL-NDT80 GAL4.ER cells containing wild-type IME2 (UB15124) or ime2-as1 (UB16047). Cells were induced to sporulate, then treated with 1 µM β-estradiol and 20 µM 1-NA-PP1 at 5 h. Images were acquired at the time of β-estradiol addition (0 h) or 3 h later. Dashed lines: cell boundaries. Mitochondria, mitoBFP. Nuclei, Htb1-mCherry. (D and E) Quantifications for the experiment in C. Analysis was restricted to cells that had entered the meiotic divisions (n = 20 cells per group). Medians are plotted as horizontal lines. The Mann–Whitney nonparametric test was used to test statistical significance. (D) Number of Num1-GFP foci per cell (see Materials and methods). (E) Whole-cell Num1-GFP fluorescence quantification from maximum-intensity projection, normalized to cell area. (F) Localization of Num1-mKate2 and GFP-Mdm36 in live pGAL-NDT80 GAL4.ER (UB18219) and pGAL-NDT80 GAL4.ER ime2-as1 (UB18221) cells. After 5 h in SPO medium, the cultures were treated with 1 µM β-estradiol and 20 µM 1-NA-PP1. Images were acquired 4 h later. Single planes in the z-axis are shown. Entry into the meiotic divisions was staged by the prospore membrane marker BFP-Spo2051–91. Note that prospore membranes are misshapen in the ime2 mutant. (G) Maximum-intensity projections of live wild-type IME2 ndt80Δ cells (UB16806) or IME2-st ndt80Δ cells (UB16808) arrested in prophase I for 5 h. Cells express the same cellular markers as in C. (H and I) Quantifications of the experiment in G (n = 20 cells per group). Medians are plotted as horizontal lines. The Mann–Whitney nonparametric test was used to test statistical significance. (H) Number of Num1-GFP foci per cell (see Materials and methods). (I) Whole-cell Num1-GFP fluorescence quantification from maximum-intensity projection, normalized to cell area. Scale bars, 2 µm; inset panels, 400 nm.

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