Stu2 ^T866 phosphorylation is important for of anaphase spindle function. (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. Representative images of anaphase cells showing interpolar Stu2-GFP signal as dashed white line. Scale bars are 1 µm. (B) Cells grown as in A were imaged to determine interpolar Stu2-GFP signal for STU2-GFP^WT and stu2^T866V-GFP cells in anaphase. Bar represents the mean of n = 85–108 individual measurements. Error bar is SEM. P values from two-tailed unpaired t test. (C) Cells grown as in A were imaged to determine distance between Ndc80 puncta for STU2^WT-GFP and stu2^T866V-GFP cells in anaphase. Bar represents the mean of n = 85–108 individual measurements. Error bar is SEM. P values from two-tailed unpaired t test. (D) Exponentially growing stu2-AID SPC110-mCherry cells ectopically expressing STU2-GFP variants (STU2^WT-GFP, M2429; stu2^T866V-GFP, M5309) were treated with auxin for 30 min. Spc110-mCherry and Stu2-GFP signals were imaged every minute. Representative time course of a cell progressing through anaphase beginning 5 min prior to anaphase onset until 15 min postanaphase onset. The white dotted line indicates anaphase onset. Arrow indicates instance of spindle collapse/regression. Proportion of cells that showed spindle regression phenotype indicated below time courses. (E) Cells harboring stu2-AID ipl1–321 (M1995) or also expressing STU2^WT-3HA (M4627) or stu2^T866V-3HA (M4628) were serially diluted (fivefold) and spotted on plates containing DMSO (control), 500 μM auxin, or 500 μM auxin + 5 μg/ml benomyl. Plates were grown at 23°C or 34°C (i.e., semi-permissive temperature for ipl1–321). (F) WT (M3), bim1Δ (M6558), and bim1Δ stu2-AID cells with various STU2-3HA covering alleles (null, M6463; STU2^WT, M6546; stu2^T866V, M6547) were serially diluted (fivefold) and spotted on plates containing DMSO or 500 μM auxin. (G) WT (M3), kar3-AID (M6465), kar3-AID stu2-AID cells with various STU2-3HA covering alleles (null, M6467; STU2^WT, M6538; stu2^T866V, M6539) were serially diluted (fivefold) and spotted on plates containing DMSO, 500 μM auxin, or 500 μM auxin + 5 μg/ml benomyl.