Chemical Genetics of Mps1 Figure 2. Mps1 activity is essential for the mitotic checkpoint. A: Quantification of flow cytometric analysis of the fraction of mitotic cells treated with DMSO or 23dMB-PP1 alone or in combination with nocodazole for 14 hours. Graph represents average of three experiments (+/2SD). B– F: Time-lapse analysis by DIC microscopy of HCT- and UTR-derived cells treated with taxol (HCT-derived cells) or STLC (UTR-derived cells) in combination with DMSO or 23dMB-PP1. Images in B display morphology of HCT-Mps1as cells imaged at the indicated timepoints after addition of taxol in combination with DMSO or 23dMB-PP1. Graphs in C and E represent percentages of cells delayed in mitosis for 4 hours or longer. Percentages are indicated at top of each column. Line graphs in D and F show cumulative percentages of cells that display first signs of furrowing at indicated times after NEB. Dashed lines indicate half-time to furrowing of inhibited drug-treated Mps1as cells. doi:10.1371/journal.pone.0010251.g002 (Fig. 4A). Similarly, Mad2 immunoprecipitations displayed little bound Cdc20 in cells treated with 23dMB-PP1 when compared to DMSO-treated cells (Fig. 4B). These data show that Mps1 catalytic activity is needed for formation and/or stability of MCC. Mps1 inhibition decreases the time spent in mitosis To get more detailed insight in the functions of Mps1 kinase activity during mitosis, we examined various aspects of chromo- some segregation in our cell lines by time-lapse microscopy. Mps1WT cells treated with DMSO or 23dMB-PP1 progressed through mitosis normally. In addition, 23dMB-PP1 treatment of Mps1as-expressing cells caused no overt problems in mitotic entry, bipolar spindle formation or cytokinesis in neither cell line. Strikingly, however, 23dMB-PP1-treated Mps1as cells sped through mitosis (Fig. 5A). To more carefully quantify this phenotype, the time from NEB to the first signs of furrowing PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e10251