Sad1 accumulation at the SPB is promoted by telomeres and centromeres. (A) Mean Sad1-GFP signal intensities at the SPB through meiosis were quantified in 19 WT meiocytes, 21 bqt1Δ meiocytes showing spindle defects, and 19 bqt1Δ meiocytes with proper spindle formation. The data shown are from >10 independent experiments. 0 min represents the onset of MI and error bars represent standard deviations. Green shading indicates the period of horsetail SPB movement. (B) Sad1-GFP/Sid4-mCherry intensity ratios are shown for the same cells quantified in A, as Sid4 intensity profiles through meiosis are identical in WT and bouquet-defective meiocytes (unpublished data). (C, left) Schematic of the strategy used to achieve an approximate halving of Sad1 level. A diploid constructed by crossing h⁺ sad1-GFP hht1-mRFP and h⁻ sad1-A323V-GFP hht1-mRFP was meiotically induced. Once prophase horsetail movement commenced, the temperature was switched to 32°C to inactivate Sad1-A323V; the subsequent meiosis was filmed to assess spindle formation. (right) Sad1-GFP intensities, from 10 meiocytes for each genotype shown, are plotted over time relative to MI onset. Yellow shading indicates the time points taken at 25°C; the subsequent time points were taken 32°C. (D and E) Series of frames of Sad1-GFP/Sad1A323V-GFP meiocytes showing SPB problems when the temperature was switched to 32°C. Bars: (black) 5 µm; (gray) 1 µm. (F) Quantitation of Sad1-mCherry intensity levels in the strains indicated. Greater than 10 meiocytes are represented for each genotype.