Figure 3.
Co-expression of non-degradable Sgo1 and Mps1 in meiosis II does not interfere with silencing of the SAC. (A and B) Control (z26045) and PEST-sgo1-mD PEST-mps1-mD (z26015) cells containing the CDC20-mAR system were transferred to sporulation medium (SPM, t = 0) and released from the metaphase I arrest with CuSO4 at t = 480 min (arrow heads). Estradiol was added at t = 495 min. Note that endogenous and non-degradable Sgo1 carry Myc9 tags while both versions of Mps1 are tagged with Ha3.43 (A) Top, immunoblot detection of proteins in whole-cell extracts.44 Cyc, samples from proliferating cells. Bottom, percentages of cells with one (2 nuclei) or 2 divisions (more than 2 nuclei), meiosis I or meiosis II spindles, and spheres stained by FITC-concanavalin A (Con A spheres) were determined by immunofluorescence microscopy of fixed cells.14 Sgo1 foci were detected on chromatin spreads.44 100 cells were counted per time point. (B) Cells stained with FITC-concanavalin A and DAPI at t = 640 min. Spheres around the nuclei represent mannoproteins in the prospore membrane and the first (inner) spore wall layer.61 Scale bar = 2 μm.