(A) HeLa cells were transfected with plasmids to express mCherry-Ska1, mCherry-PP1, mCherry-Ska1ΔCTD, and mCherry-Ska1ΔCTD fused to PP1 (mCherry-Ska1ΔCTDPP1). Endogenous Ska1 was depleted using Ska1 siRNA. Hoechst 33342 was added at 25 ng/ml to visualize DNA. Cells were then imaged by time-lapse microscopy, and % of mitotic cells arrested in metaphase was plotted. As expected, expression of siRNA-resistant mCherry-Ska1 rescued metaphase arrest caused by Ska1 depletion while expression of mCherry-PP1 or mCherry-Ska1ΔCTD did not. Expression of Ska1ΔCTDPP1 fusion in Ska1 depleted cells completely rescued metaphase arrest. (B) The interval from NEB to anaphase onset is plotted for cells that progressed to anaphase while expressing Ska1 constructs without or with depletion of endogenous Ska1. As expected, Ska1ΔCTD-expression showed a dominant negative effect delaying mitotic progression in control cells not depleted of endogenous Ska1. Expression of the fusion, Ska1ΔCTDPP1, caused no delay. When endogenous Ska1 was depleted, 38% of cells arrested at metaphase and did not progress to anaphase (Figure 7A). The rest showed delayed progression from NEB to anaphase with an average of 110 min compared to control cells (35 min). As expected, the delay was rescued by expression of mCherry-Ska1 but was not rescued by expression of mCherry-PP1 or mCherry-Ska1ΔCTD. Expression of Ska1ΔCTDPP1 showed significant rescue of the delay cause by Ska1 depletion with an average time from NEB to anaphase of 62 min. (C) HeLa cells were transfected with the indicated plasmids and then treated with mock or Ska1 siRNA. Expression of Ska1ΔCTD fused to a phosphatase dead PP1 (Ska1ΔCTDpdPP1) failed to rescue Ska1 depletion. Indeed, expression of phosphatase dead fusion, on its own, induced a potent metaphase arrest phenotype in cells not depleted of endogenous Ska1. Moreover, it exacerbated the metaphase arrest in cells depleted of Ska1. (D) Ska1ΔCTDpdPP1 causes a longer delay to anaphase onset than Ska1ΔCTD even without depletion of endogenous Ska1. (E) Chromosome alignment is delayed in Ska1 depleted cells and this is recapitulated by expression of Ska1ΔCTDpdPP1 without endogenous Ska1 depletion. Delays in chromosome alignment are not observed upon expression of Ska1ΔCTDPP1 fusion. (F) Hypothetical model for dynamic balance of Mps1 kinase and PP1 phosphatase activities during mitotic progression. Photobleaching studies have shown that Mps1, Ska, and PP1 all exhibit high turnover at kinetochores with a residence times of a few seconds (Raaijmakers et al., 2009; Howell et al., 2004; Trinkle-Mulcahy et al., 2003). Before microtubule attachment, Mps1 concentration at kinetochores remains high due to interaction with the CH domains of the Ndc80 complex. Correspondingly, Ska-PP1 concentrations are low because of the paucity of microtubules. The high Mps1 and low PP1 concentrations maintain high phosphorylation Mps1 substrates, Knl1 and Bub1. Microtubules compete with Mps1 for binding to the CH domains of the Ndc80 complex, resulting in depletion of Mps1. The binding of Ska to microtubule protofilaments increases PP1 concentration. High Ska-PP1 and low Mps1 result in dephosphorylation of substrates, promoting release of Bub1–Bub3 and Mad1–Mad2 complexes. Diminished checkpoint signaling due to release of Bub1–Bub3 and Mad1–Mad2 from kinetochores promotes anaphase onset and mitotic exit.