he tension checkpoint response. In contrast, though, the tension checkpoint is rendered ineffective by mimicking constitutive Sli15 phosphorylation by Ipl1. Strain wt sli15-20A sli15-20D Red colonies 17 48 114 Half-sectored colonies 6 55 75 Total colonies 4160 4140 5088 Chromosome loss rate per 
cell division a 1.45 13.4 15.1 Fold increase 9.3 10.4 a Calculated as 4. doi:10.1371/journal.pone.0089399.t005 9 Ipl1-Dependent Phosphorylation of Sli15 Sli15-20D was virtually undetectable even at the highest protein concentration used. Thus the in vitro microtubule binding properties of the recombinant Sli15 proteins mirrored their behavior in vivo in metaphase-arrested cells and imply that the addition of multiple, negatively charged groups to the Sli15 microtubule-binding domain disrupts its ability to bind microtubules. To confirm that the multiple amino acid substitutions in Sli1520D were mimicking phosphorylation of Sli15 by Ipl1, we next compared the microtubule binding properties of GST-Sli15 in the presence of Ipl1, after pre-incubation either alone or with addition of ATP to allow Sli15 phosphorylation. indicating that phosphorylation of Sli15 by Ipl1 suppresses its affinity for microtubules. However, the phosphorylated complex still showed significant binding in comparison to Sli15-20D, possibly because the stoichiometry of phosphorylation was not maximal or because of additional effects of the Sli15-20D mutations. Sli15-20A and Sli15-20D show opposite patterns of localization along the anaphase spindle We next examined the localization of the three Sli15 proteins in cells allowed to progress from metaphase to anaphase. In anaphase cells expressing wild-type Sli15-EGFP the protein decorated the full length of the extended anaphase spindle in a punctate manner. However, in the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19638617 equivalent sli15-20A strain, although Sli15 was clearly localized along the spindle, it frequently appeared more abundant in the central region. In contrast, 10 Ipl1-Dependent Phosphorylation of Sli15 Sli15-20D towards the poles of the anaphase spindle is reminiscent of the pattern seen when Sli15 remains phosphorylated on its Cdk sites but cannot be phosphorylated on its Ipl1 sites. Thus phosphorylation of Sli15 may both reduce overall microtubule association and bias Sli15 localization towards the poles and away from the mid-zone of the anaphase spindle, with intermediate levels of phosphorylation on either the Ipl1 or Cdk sites being insufficient to prevent association. Strains lacking three distinct mechanisms for regulating PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639073 the interaction of the CPC with spindle microtubules fail to show an additive growth defect Recent work has indicated that several mechanisms influence the interaction of yeast CPC with microtubules including a weak, direct interaction between microtubules and Ipl1 itself that we have confirmed in our work, binding via Bim1 that is antagonized by Cdc28 phosphorylation on Ser-50 and Ser-76 in Ipl1, Cdc28 phosphorylation of Sli15 that is antagonized by Cdc14 phosphatase as cells enter anaphase and phosphorylation of Sli15 by Ipl1 as shown here. Given the highly conserved Neuromedin N nature of CPC relocalization to the spindle in anaphase, interfering with each of these pathways individually has surprisingly little impact on cell viability or proliferation, indicating that these mechanisms might function in a redundant manner to regulate CPC localization. We therefore attempted to generate strains containing different combinations of either alanine or p
