Ipl1 phosphorylation negatively regulates She1 spindle localization and MT cross-linking activity. (A) Percentage of metaphase-arrested spindles exhibiting the bent phenotype in WT, she1Δ, and she1-ΔN126 cells treated with DMSO (−) or 50 µM 1-NA-PP1 (+). Error bars are standard error of proportion (n ≥ 50 for each). Inset, sensitivity of ipl1-as5 to 1-NA-PP1. (B) Localization of EGFP-tagged She1_WT, She1_5A, and She1_5D in Cdc20-depleted mRuby2-Tub1 cells. (C) Quantification of spindle-associated EGFP-She1_5A and She1_5D relative to She1_WT. Error bars are SEM (n ≥ 70 spindles for each). P < 0.0001 by unpaired t test. (D) Quantification of the bent spindle phenotype in She1_5A and She1_5D relative to She1_WT. Error bars are standard error of proportion (n ≥ 80 cells each, P < 0.01 by t test). (E) Representative video frames of metaphase spindles for quantification in D. (F) She1_5D purified from Escherichia coli. (Right) Intensity profiles for 142 cross sections of MTs (gray lines). Inset, representative image. Black line = mean intensity. Mean intensity for She1-FL (orange dashed line) from Fig. 3 C is overlaid for comparison. (G) Colocalization correlation analysis of Ipl1-3GFP with Sli15-mRuby2 or mRuby2-She1 along a WT anaphase spindle. Yellow arrows indicate Ipl1-3GFP puncta, whereas arrowheads indicate Sli15-mRuby2 (top) or mRuby2-She1 (bottom) puncta. Error bars are SEM. (H) Model showing She1 function on the metaphase spindle. We propose that She1 cross-links ipMTs and prevents premature loading of Ipl1 (see Discussion). In she1-ΔN126 cells, Ipl1 prematurely loads onto the metaphase spindle, causing a decrease in the levels of both She1 and Bim1, resulting in the bent spindle phenotype and increased ipMT dynamics. kMTs, kinetochore MTs. SPB, spindle pole body.