PP2A ^Cdc55 regulation of mitotic spindle and model of phosphoregulation of Stu2 subcellular localization and function. (A) Model of PP2A^Cdc55 substrates involved in spindle regulation detected by mass spectrometry (see Fig. S5 G). Putative activity of Cdc5/Plk1 indicated by question mark. (B) Exponentially growing SPC110-mCherry MTW1-3GFP cells containing CDC55^WT (M1174) or cdc55Δ (M5848) were imaged every minute. Spindle length of cells in metaphase was determined by measuring the distance between Spc110-mCherry foci. Each data point represents an individual cell. Bars represent the average of n = 20–23 individual measurements. Error bars are SEM. P values from two tailed unpaired t test. (C) Cells grown and imaged as in B were analyzed to determine maximum rates of spindle elongation over a 2-min period for each individual cell. Each data point represents a single cell. Bars represent the average of n = 20–23 individual measurements. Error bars are SEM. P values from two-tailed unpaired t test. (D) Model of phosphoregulation of Stu2 localization. In G1 phase, Stu2 is predominantly localized to the cytoplasm. As Cdk activity increases in the cell cycle, Stu2^S603 becomes phosphorylated, and Stu2 is imported into the nucleus. In the nucleus, Stu2 binds to many factors, including Ndc80c (as well as +TIP factor Bik1, Kip3, Kar9, and others), and carries out a required function for chromosome biorientation. During metaphase, Plk1/Cdc5 interacts with Stu2 via binding phosphorylated Stu2^S603. Plk1 phosphorylates Stu2^T866, and PP2A^Cdc55 opposes this phosphorylation. At anaphase onset, PP2A^Cdc55 activity is inhibited by separase, and Stu2^T866 becomes predominantly phosphorylated. This leads to reduced Stu2:Ndc80c association and relocalization of some Stu2 to spindle microtubules to regulate anaphase spindle function. Stu2 localization at spindle-pole bodies (via Spc72) is also indicated. Reduced association with +TIP factor Bik1 may also be possible upon Stu2^T866 phosphorylation.