Stu2 ^T866 phosphorylation is opposed by PP2A ^Cdc55 until anaphase onset. (A) Exponentially growing stu2-AID NDC80-mKate cells expressing STU2-GFP (M3774) or stu2^T866V-GFP (M4429) were released from a G1 arrest into auxin-containing media. Samples were taken every 15 min, fixed, and imaged. Left: Representative images of mitotic cells with pre-anaphase–like Ndc80 distance (short, <1.5 µm) and anaphase-like Ndc80 distance (long, >1.5 µm). Scale bars are 1 µm. The dotted line indicates how Ndc80 distance was measured. Right: Kinetochore-proximal Stu2-GFP signal and Ndc80-mKate signal were measured for individual puncta. Ratio of Stu2/Ndc80c signal plotted for cells with short (<1.5 µm) or long (>1.5 µm) Ndc80 distance. Measurements are an average from two replicate time-course experiments. Bars represent the mean of n = 212–492 individual measurements. Error bars are SEM. P values from unpaired t tests. Note long Ndc80 distance panels also appear in Fig. 1 A. (B) Cells grown and imaged as in A. Ratio of Stu2/Ndc80c signal was plotted for cells binned together by Ndc80 distance (0 µm: 0 µm ≤ × ≤ 1.5 µm; 1.5 µm: 1.5 µm < × ≤ 3 µm, etc.). Data points are means of many individual measurements (STU2^WT: 0 µm n = 337, 1.5 µm n = 345, 3.0 µm n = 42, 4.5 µm n = 111, 6.0 µm n = 108, 7.5 µm n = 104, 9.0 µm n = 110. stu2^T866V: 0 µm n = 274, 1.5 µm n = 182, 3.0 µm n = 63, 4.5 µm n = 48, 6.0 µm n = 58, 7.5 µm n = 50, 9.0 µm n = 38). Error bars are SEM. (C) Exponentially growing stu2-AID cdc20-AID cells with an ectopically expressed STU2-GFP allele (M5335) and stu2-AID cdc20-AID cdc55Δ cells with ectopic STU2^WT-GFP (M5337) or stu2^T866V-GFP (M5611) were cultured in auxin-containing media to arrest cells in metaphase. Cells were fixed and imaged to determine kinetochore-proximal Stu2-GFP. Bars represent the average of n = 98–104 individual measurements. Error bars are SEM. P values from two-tailed unpaired t tests. (D) Model of PP2A^Cdc55 opposing Cdc5 activity against Stu2^T866. Left: In metaphase, PP2A^Cdc55 is highly active and removes phosphorylation from Stu2^T866; this cycling results in a steady-state low level of pT866. Right: In anaphase, separase inhibits PP2A^Cdc55, allowing Cdc5 to win and resulting in rapid net phosphorylation of Stu2^T866.