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Figure S3. Mad2 localization, its functional relationship with APC/C activators, and MII spindle dynamics in the rec12 mutant. (A) The yeast two-hybrid analysis of Mad2 interaction with APC/C activators. A genomic DNA fragment encoding a full-length gene of slp1⁺ was amplified by PCR using the genomic DNA as a template and cloned into pGBD-C1 (James, P., J. Halladay, and E.A. Craig. 1996. Genetics. 144:1425–1436). The cDNA fragments encoding full-length genes of fzr1⁺ and mad2⁺ were amplified by PCR using the Schizosaccharomyces pombe cDNA library as a template and cloned into the pGBD-C1 and pACT2 plasmids (Clontech Laboratories, Inc.), respectively. A budding yeast strain containing the plasmids was grown on selective (−Ade and −His) and nonselective (+Ade and +His) media. AD, plasmids expressing a GAL activation domain; BD, plasmids expressing a GAL DNA-binding domain. (B and C) Intracellular localization of Mad2-GFP at meiosis in the wild type (B) and rec12 mutant (C). Bottom images in B indicate colocalization of Mad2-GFP dots with Nuf2-vizualized centromeres. Bottom enlarged images in C show prolonged centromere localization of Mad2-GFP at MI in the rec12 mutant (arrowheads). At MII, Mad2-GFP was localized to the centromere and/or the SPB before anaphase and disappeared at anaphase in both types of cells (Meiosis II). The SPB is simultaneously visualized by Sad1 tagged with DsRed (magenta). Numbers indicate time in minutes. (D) Effects of mitotic expression of APC/C activators on the SAC-induced metaphase arrest. An slp1⁺ genomic DNA fragment or an fzr1⁺ cDNA fragment was cloned into a pREP1 plasmid (pREP1-Slp1 or pREP1-Fzr1) such that the expression was driven by the thiamine-repressible nmt1 promoter. The nda3 mutant cells containing a pREP1-slp1+ (Slp1), pREP1-fzr1⁺ (Fzr1), or pREP1 (Vector) were grown in the liquid EMM medium containing 10 µM thiamine at 30°C to the logarithmic phase and then transferred to YPD medium. After incubation for 4 h at 30°C, the cells were further incubated for 18 h at 18°C to induce the metaphase arrest. Experiments were performed under conditions in which the expression was low because the forced expression of slp1⁺ or fzr1⁺ was lethal. The cells were fixed by 3.7% formaldehyde and stained for DNA with DAPI. The cells were then scored for chromosome morphology. Black and white bars indicate cells with condensed and noncondensed chromosomes, respectively. The graph shows results of at least two independent experiments. (E) MII spindle behavior in the rec12 mutant. (right) Graph shows changes in the spindle length. The broken lines show the boundaries of the spindle phases. (F) Distribution of MII spindle lengths. Each plot shows maximum lengths of the two MII spindles (spindle 1 and spindle 2) in the same cell. Unlike the two spindles in a wild-type cell, those in a rec12 cell were markedly different in length at MII. The broken lines indicate two spindles with equal lengths. (G) MII spindles and chromosome masses. Error bars indicate standard deviation. Bars, 5 µm.