chromosomes, causing frequent but non-lethal chromosome segregation errors [16]. Conversely, reduction of Mps1 activity has recently been shown to sensitize tumor cells but not normal cells to low doses of taxol by elevating the frequency of chromosome missegregations to near-lethal levels [17]. Partial inhibition of Mps1 might therefore be an effective anti-cancer therapy. Although RNAi studies have uncovered several aspects of human Mps1 biology, the multifunctional character of Mps1 has prevented detailed and temporally controlled investigations into the different roles Mps1 might play in mitosis. Inhibition using the small molecules SP600125 and cincreasin has proved to be useful [18,19], but cincreasin does not inhibit Mps1 in human cells [19] and the non-specific nature of SP600125 makes it an unfavorable choice to study Mps1. A more controlled approach is the use of chemical genetics, in which endogenous kinase is replaced by an engineered protein containing a mutated gatekeeper residue [20]. These gatekeeper mutants render the kinase specifically sensitive to inhibition by non-hydrolysable bulky ATP analogs such as chemically modified variants of the Src inhibitor PP1. This approach has previously been described for Mps1 in budding yeast [6,13] and in human cells in combination with transient RNAi [21]. The use of transient RNAi, however, introduces uncertainties regarding efficiency of knock down and reproducibility. We present two cell lines in which endogenous Mps1 has been stably replaced with gatekeeper mutants. In-depth analysis of these clonal cell lines showed that addition of bulky PP1 analogs allowed rapid and reversible inhibition of Mps1 kinase activity in a highly penetrant and reproducible manner. Using these cell lines we dissected the different roles of Mps1 in mitosis, further establishing known functions in checkpoint regulation and error-correction and providing novel insights in the function of Mps1 catalytic activity in checkpoint regulation. Results Mps1 is selectively inhibitable in two engineered cell lines To study the role of Mps1 in different mitotic processes, we created cell lines in which Mps1 kinase activity could specifically and reversibly be inhibited. For this, we engineered the ATP- binding pocket of Mps1 to create a kinase with unique preference for bulky ATP-like small molecules [20]. As previously shown for Mps1 in a transient expression system [21], Mps1M602A signifies such an analog sensitive (as) version. In addition to M602A, we also tested M602G, which similarly enlarges the ATP-binding pocket of Mps1 and may provide stronger inhibition [6]. To ensure that mutation of the gatekeeper residue did not affect Mps1 functionality, the ability of both Mps1M602A and Mps1M602G to sustain an active checkpoint in Mps1 RNAi cells was investigated. Similar to LAP-MpsWT, both LAP-Mps1M602A and LAP- Mps1M602G reconstituted checkpoint signaling in response to nocodazole when endogenous Mps1 was transiently replaced with these proteins (Fig. S1A). To create stable cell systems in which Mps1 could be inhibited in a potent and reproducible manner, we stably expressed LAP- Mps1M602A (hereafter referred to as Mps1as) or LAP-Mps1M602G in UTRM10 (U2OS-derived) and HCT-TRM (HCT116-derived) cell lines in which endogenous Mps1 could be removed by doxycycline-induced expression of Mps1 shRNA [17]. Continuous growth in doxycycline and clonal selection resulted in stable, viable clonal cell lines in which endogenous Mps1 was undetectable and replaced with Mps1WT or Mps1as (Fig. 1A). No HCT-TRM clones expressing Mps1M602G were obtained and only one such Chemical Genetics of Mps1 UTRM10 clone grew out. We suspect that the low activity of Mps1M602G (see below) significantly decreased the chance of survival of cells expressing only this form of Mps1. Nevertheless, one UTRM10 cell line expressing Mps1M602G was established, showing that under certain circumstances, Mps1M602G can support viability. All cell clones (renamed HCT-Mps1WT, HCT- Mps1as, UTR-Mps1WT, UTR-Mps1as and UTR-Mps1M602G) have been kept in culture for months, showing that replacement of Mps1 is stable. Human Mps1 auto-activates by phosphorylating T676 in its activation loop. T676 phosphorylation is essential for full kinase activity [16,22,23], and the phosphorylation status of T676 serves as a read-out for Mps1 kinase activity [16]. To investigate if Mps1as activity could be inhibited by bulky PP1 analogs in our engineered cell lines, we examined Mps1 T676 phosphorylation in different conditions. Following tests on the potency of different PP1 analogs to inhibit Mps1as (Fig. S1B), we settled on 23dMB- PP1 (1-(tert-butyl)-3-(2,3-dimethylbenzyl)-1H-pyrazolo[3,4-d]pyri- midin-4-amine) as the Mps1as inhibitor of choice. As expected, Mps1WT and Mps1as displayed a mobility shift and phosphory- lation of T676 upon nocodazole addition in both HCT and UTR cell types (Fig. 1B). Whereas addition of 23dMB-PP1 did not affect the mobility or T676 phosphorylation of Mps1WT, 23dMB-PP1 prevented hyperphosphorylation as well as T676 phosphorylation of Mps1as (Fig. 1B). Full inhibition of Mps1as was established within 5 minutes after addition of 23dMB-PP1 (Fig. 1C). Further- more, T676 phosphorylation could be detected 15 minutes after removal of 23dMB-PP1, showing that this rapid inhibition was reversible (Fig. 1D). Interestingly, very little T676 phosphorylation and weak mobility shift was detected in the UTR-Mps1M602G clone, indicating this engineered Mps1 kinase has strongly reduced activity compared to Mps1WT and Mps1as (Fig. S2A). Although any residual activity and all Mps1 functions could be efficiently inhibited by treatment of the UTR-Mps1M602G cells with PP1 inhibitors (Fig. S2 and S3B), we focused our investigations on studies with the Mps1as clones. Mps1as inhibition by 23dMB-PP1 disables mitotic checkpoint signaling Mps1 kinase activity is needed for the mitotic checkpoint to delay mitosis when unattached kinetochores persist [6,9,12,18,21]. We next studied the ability of the cell lines to maintain a mitotic checkpoint response in the presence of 23dMB-PP1. In absence of 23dMB-PP1, all HCT-Mps1WT and HCT-Mps1as cell lines sustained checkpoint activity in the presence of nocodazole as scored by mitotic index (Fig. 2A). The ability to maintain a nocodazole-induced mitotic delay was not lost in Mps1WT cells upon treatment with 23dMB-PP1, showing 23dMB-PP1 did not have off-target effects that could disable the mitotic checkpoint. In contrast to Mps1WT-expressing cells, 23dMB-PP1 completely abolished mitotic checkpoint activity in Mps1as cells. Similar results were obtained with the UTR-Mps1M602G cells (Fig. S2B). To ensure that Mps1 inhibition decreased mitotic index by inhibiting mitotic checkpoint activity rather than, for instance, preventing entry into mitosis, mitotic progression in the presence of spindle poisons was monitored by live cell differential interference contrast (DIC) microscopy. As a measure for time spent in mitosis, the time from nuclear envelope breakdown (NEB) to the first signs of furrow ingression was scored. Engaging the mitotic checkpoint in these cells by treatment with the spindle drugs nocodazole or taxol, or the Eg5 inhibitor STLC caused all cell lines to delay mitosis for hours (Fig. 2B–F and S3). Delays were most pronounced in cells with the HCT-TRM background where ,95% of cells maintained the arrest for at least 4 hours (Fig. 2C, PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10251