Chemical Genetics of Mps1 Figure 6. Mps1 activity is required for efficient correction of erroneous attachments. A: Chromosome alignment in HCT- and UTR-Mps1WT and -Mps1as cell lines treated with MG132 for 60 minutes in combination with DMSO or 23dMB-PP1. Graphs display percentages of cells with misaligned chromosomes, averages of three experiments, at least 50 mitotic cells per experiment (+/2SD). B, C: Immunostainings of HCT-Mps1WT and HCT-Mps1as cells treated with monastrol for 30 min followed by a release from monastrol into MG132 with or without 23dMB-PP1 for 50 minutes. Cells in B were immunostained for tubulin (green) and centromeres (ACA, red). DNA (DAPI) is in blue. Graph in C indicates the percentage of mitotic cells with misaligned chromosomes 50 minutes after monastrol washout, averages of two experiments, at least 100 mitotic cells per experiment (+/2SD). D: Immunoblots of Borealin after phosphate-affinity gel electrophoresis of lysates from nocodazole-treated HCT-Mps1as cells to which DMSO or 23dMB-PP1 and MG132 was added during the final hour. E, F: Immunoblots of the indicated proteins in lysates of the UTR- (E) and HCT-Mps1 (F) variant cell lines. Cells were treated either with thymidine (thy), with taxol for 14 hours followed by the indicated inhibitors for an additional 2hrs, or with the indicated inhibitors for 1 hour followed by taxol/MG132 treatment for an additional 2 hrs ZM, ZM447439. Arrow in F points to Cyclin B. Asteriks indicates aspecific band in thymidine sample. doi:10.1371/journal.pone.0010251.g006 Discussion In the present study we investigated the different roles of Mps1 in mitosis of human cells through the use of chemical genetics. We report here two clonal cell lines expressing analog-sensitive versions of Mps1 in which endogenous Mps1 was stably removed by doxycycline-induced shRNA expression. Use of these cell lines circumvents experimental limitation in reproductivity and pene- trance associated with transient RNAi reconstitutions [21] and allows temporal control of Mps1 activity. Chemical genetic inhibition of Mps1 was previously used to uncover novel aspects of Mps1 in S. cerevisiae mitosis [6,13]. A yeast strain carrying the analog-sensitive M516G mutation (similar to the M602G mutation in human Mps1, included in the present study) allowed analysis of Mps1 functions after the moment of Figure 7. Specific inhibition of Mps1 enzymatic activity causes cell death. HCT-Mps1WT and UTR-Mps1WT cells (A) and HCT-Mps1as and UTR-Mps1as cell (B) were treated with or without (-) 23dMB-PP1 for 8 days (8d), or with 23dMB-PP1 for 2 days (2d) followed by wash-out and continued growth until day 8. doi:10.1371/journal.pone.0010251.g007 spindle pole body duplication, a critical function of Mps1 in budding yeast [4,6]. Using this strain (mps1-as1), it was verified that Mps1 inhibition prevents mitotic checkpoint activity and causes cell death [6]. In addition, Mps1-inhibited yeast cells were found to display spindle morphology defects and chromosome misposi- tioning [6], possibly as a consequence of errors in chromosome biorientation [13]. In agreement with these and other reports [6,12,13], chemical genetic inhibition of Mps1 in our human cell lines verified that Mps1 kinase activity is critical for chromosome biorientation. Although our data using Mps1 RNAi supported the hypothesis that Mps1 facilitated error-correction by promoting Aurora B activity at inner centromeres [12], we now show that error- correction is also prevented when Mps1 kinase activity is inhibited only during the error-correction process in cells undergoing spindle bipolarisation after monastrol wash-out. Additionally, through the use of phosphate-affinity gel electrophoresis we show that phosphorylation of endogenous Borealin is under the control of Mps1. Borealin phosphorylation enhances Aurora B activity [12,14] yet inhibition of Mps1 did not affect pS10-H3 levels under conditions that showed an increase in Borealin mobility. pS10-H3 was only found reduced when cells entered mitosis without Mps1 activity. These results suggest that partial reduction in Aurora B activity caused by Mps1 inhibition is difficult to detect once pS10- H3 is fully established, possibly because of low turnover of the phosphate group on S10-H3. In support of this, inactivation of Aurora B by a 2 hour exposure of our HCT-derived cells to ZM447439 only caused a moderate reduction of pS10-H3 levels. These data indicate that pS10-H3 serves as a good readout for Mps1-dependent regulation of Aurora B activity when pS10-H3 is not fully established at mitotic entry. Nevertheless, since Mps1 inhibition during mitosis affects Borealin phosphorylation and error-correction, it is likely that acute effects on Aurora B activity by Mps1 inhibition can be visualised with antibodies to the relevant error-correction substrates. The function of Mps1 kinase activity in the regulation of the mitotic checkpoint has previously been established by various labs including ours, and the present data fully agree with this [6,9,12,18,21]. As expected [11,12,24], Mps1 inhibition in our cell lines caused mislocalization of Mad1 and Mad2. This disagrees with the study by Tighe et al., in which chemical genetic inhibition of Mps1 in cells transiently transfected with Mps1 shRNA plasmids had little effect on Mad1 recruitment [21]. This discrepancy may be due to differences in penetrance of Mps1 inhibition. Whereas inhibition of Mps1 with 1NM-PP1 in the Tighe et al. study left mitotic progression unaffected in a large proportion of cells, mitosis was disturbed in the vast majority (UTR-Mps1as) or all (HCT- Mps1as) cells in our cell lines (Fig. 2 and 5). This difference in penetrance may be due to inefficient RNAi by transient shRNA transfection or potency of 1NM-PP1 in the Tighe et al., study. In our hands, 1NM-PP1 was significantly less capable of inhibiting Mps1 than 23dMB-PP1 or 3MB-PP1 (Fig. S2). Besides Mad1/ PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e10251