MECA is dynamically regulated in meiosis. (A) Left: diagram of the mitochondria–plasma membrane contact site. MECA contains two known subunits, Num1 and Mdm36, and is responsible for tethering mitochondria to the plasma membrane. Right: localization of MECA subunits, Num1-mKate2 and GFP-Mdm36, in live premeiotic and meiosis II cells (UB16677). Single planes in the z-axis are shown. The meiotic stage was determined by the prospore membrane marker BFP-Spo2051–91. (B) Maximum-intensity projections of live pGAL-NDT80 GAL4.ER cells (UB15124) showing MECA localization (Num1-GFP), mitochondrial localization (mitoBFP), and nuclei (Htb1-mCherry). Cells were imaged at time of transfer to SPO medium (premeiotic) and 5 h (prophase I). Then, at 5 h, the culture was split and treated with 1 µM β-estradiol (+Ndt80) or ethanol vehicle control (−Ndt80). Cells from the split cultures were imaged at 8 h. Dashed lines: cell boundaries. (C and D) Quantifications of the experiment in B. Analysis of the +Ndt80 sample was restricted to meiosis II stage cells (n = 20 cells per group). Medians are plotted as horizontal lines. The Mann–Whitney nonparametric test was used to test statistical significance. (C) Number of Num1-GFP foci per cell (see Materials and methods). (D) Whole-cell Num1-GFP fluorescence quantification from maximum-intensity projection, normalized to cell area. (E and F) Quantifications of Num1 and Mdm36 steady-state protein levels (MS) and synthesis (ribosome profiling) during meiosis from a published dataset (Cheng et al., 2018). (G and H) Immunoblots of MECA subunits in pGAL-NDT80 GAL4.ER strains. Strains were induced to sporulate for 5 h, then flasks were split and treated with 1 µM β-estradiol (+Ndt80) or ethanol vehicle control (−Ndt80). Hxk2 serves as a loading control. (G) Blot for Num1-3V5 (UB12402). Num1 runs above the highest (190-kD) ladder band. (H) Blot for Mdm36-3V5 (UB13851). Scale bars, 2 µm; inset panels, 400 nm.
