Chemical Genetics of Mps1 Figure 4. Mps1 inhibition reduces Mad2-Cdc20 interaction. A, B: Immunoblots of the indicated proteins in immunoprecipitations of Cdc20 (A) or Mad2 (B). Immunoprecipitations were preformed on interphase cell lysates (thymidine, thy) or mitotic cell lysates (nocodazole, noco) of HCT- Mps1as cells treated with DMSO or 23dMB-PP1 in combination with MG132 for 1 hour. Quantification of Mad2 levels in Cdc20 immunoprecipitates, relative to interphase levels, is indicated in A, right panel. doi:10.1371/journal.pone.0010251.g004 timeframe as these unperturbed mitosis: 13 vs. 10 minutes, respectively (Fig. 2D). A similar, albeit less consistent effect was apparent in the UTR-derived cell lines: Half-time to furrowing in UTR-Mps1as cells was reduced from 18 to 12 minutes by treatment with 23dMB-PP1, whereas this was not altered by 23dMB-PP1 in UTR-Mps1WT cells (Fig. 5C). Mps1 activity facilitates attachment error-correction Mps1 is critical for chromosome biorientation in yeast and humans [12,13] and our previous results using Mps1 RNAi supported the hypothesis that Mps1 facilitates error-correction by promoting Aurora B activity at inner centromeres [12]. Never- theless, a true display of the role for Mps1 in error-correction requires inhibition of Mps1 activity solely during the error- correction process. Our analog-sensitive cell lines provided the opportunity to test this. We first verified that Mps1 inhibition prevented efficient chromosome alignment on the cell’s equator. Cells were treated with MG132 for 60 minutes to prevent exit from mitosis and provide cells with sufficient time to align all chromosomes. Roughly 30% of DMSO- or 23dMB-PP1-treated HCT-Mps1WT cells or DMSO-treated HCT-Mps1as cells dis- played misaligned chromosomes within this time (Fig. 6A). In agreement with our previous studies [12], addition of 23dMB-PP1 to HCT-Mps1as cells caused severe alignment problems in ,70% of cells. Similar results were obtained by Mps1 inhibition in UTR- Mps1M602G cells (Fig S2C). We next investigated if the generated cells lines were able to correct faulty attachments when Mps1 was inhibited during error-correction rather than prior to the establishment of errors. To this end, cells were treated with the Eg5-inhibitor monastrol to create monopolar spindles with large numbers of chromo- somes that have syntelic or monotelic attachments [29]. Monastrol can efficiently be removed from cell, which allows the formation of a bipolar spindle in which full chromosome alignment requires correction of the improper attachments by Aurora B [30,31]. All attachment errors were corrected and full chromosome alignment was restored in 87% of HCT-Mps1WT cells 50 minutes after removal of monastrol (Fig. 6B, C). Addition of 23dMB-PP1 during monastrol washout of Mps1as cells however, caused significant problems with chromosome alignment (Fig. 6B, C). Importantly, the non-aligned chromo- somes were often seen to have retained syntelic attachments, indicative of error-correction deficiency. These data solidify the notion that Mps1 activity is needed for efficient correction of erroneous attachments. Mps1 enhances Aurora B activity Erroneous kinetochore-microtubule attachments cause a lack of tension between sister centromeres which allows Aurora B kinase activity to destabilize such attachments [31–36]. We and others previously found that Mps1 enhances Aurora B activity up to 4-fold by phosphorylating the CPC component Borealin, explaining the contribution of Mps1 to error-correction [12,14]. We next set out to examine the link between Mps1 and Aurora B activity in our analog-sensitive cell lines. Using phosphate-affinity gel electrophoresis [37], Borealin displayed a prominent mobility shift in mitotic samples of nocodazole- treated cells (Fig. 6D). This shift was reduced upon inhibition of Mps1as by 23DMB-PP1, showing for the first time that phosphorylation of endogenous Borealin is under control of Mps1 activity. To investigate the consequences of Mps1 inhibition on Aurora B activity, mitotic cells were collected by shake-off after 14 hours of taxol treatment and subsequently treated with 23dMB-PP1 or DMSO in combination with MG132 for an additional hour, after which mitotic lysates were examined for phosphorylation of Serine 10 in Histone H3 (pS10-H3) by immunoblot. Surprisingly, addition of 23dMB-PP1 to UTR- or HCT-Mps1as cells that had been arrested in mitosis for hours did not significantly reduce pS10-H3, even though Mps1 activity, as measured by mobility shift, was inhibited (Fig 6E, F). These findings are in seeming contrast to our previous report, in which Mps1 RNAi reduced PLoS ONE | www.plosone.org 6 April 2010 | Volume 5 | Issue 4 | e10251