Figure 4.

Abnormal behavior of Meu14-GFP in mug28Δ cells. (A) Subcellular localization of Meu14-GFP as visualized by fluorescent microscopy from meiosis II to sporulation in mug28⁺ and mug28Δ cells. YOD50 (meu14⁺-GFP) and AS160 (meu14⁺-GFP mug28Δ) cells were induced to enter meiosis by nitrogen starvation, and then Meu14-GFP (green) and DIC images were acquired by fluorescence microscopy, until sporulation occurred. Images in i–iv and v–viii represent the subcellular localization of Meu14 from meiosis II to sporulation in mug28⁺ and mug28Δ cells, respectively. Except for image viii, which was acquired at sporulation, all other Meu14-GFP images are similar between mug28Δ and mug28⁺ cells. Bar, 10 μm. (B) The histogram shows the percentage of normal and abnormal cells expressing Meu14-GFP in mug28⁺ and mug28Δ strains. (C) Typical images of abnormal mug28Δ cells expressing Meu14-GFP. Images where Meu14-GFP localized to satellite dots in the vicinity of the Meu14-GFP rings were classified as class I. White arrowheads indicate the satellite dots near the Meu14-GFP rings. Images where the Meu14-GFP signal accumulated at the leading edge of FSM even after the completion of FSM formation were classified as class II. Images where Meu14-GFP localized abnormally to uncharacterized dots in the vicinity of FSM in the cytoplasm were classified as class III. Bar, 10 μm. (D) Enlarged views of the regions indicated by white squares in C. Bar, 10 μm. (E) Meiotic expression of Meu14-GFP in pat1-114 mug28⁺ and pat1-114 mug28Δ cells. These cells were induced to enter meiosis synchronously by a temperature shift and were collected at 1-h intervals for protein extraction, blotting, and probing with the anti-GFP and anti-Cdc2 (loading control) antibodies. At each time point, the frequency of cells with one, two, three, or four nuclei was determined by counting at least 200 Hoechst33342-stained cells under a microscope (top).