Figure 1.
Stable expression of LAP-Mps1as renders selective sensitivity to 23dMB-PP1.
A: Mps1 and tubulin immunoblots of lysates of UTRM10 and HCT-TRM cells in which endogenous Mps1 is removed by doxycycline-induced expression of Mps1 shRNA and replaced with the indicated LAP-Mps1 mutants. B–D: pT676-Mps1 and Mps1 immunoblots of UTR-Mps1as (B) and HCT-Mps1as (B–D) cell lines treated with nocodazole and MG132 in combination DMSO/23dMB-PP1 for 1 hour (B), with nocodazole and MG132 for 1 hour in combination with an additional 23dMB-PP1 treatment for the indicated amounts of time (C), or with nocodazole and MG132 in combination with 23dMB-PP1 for 1 hour, followed by PBS wash after which cells were left for 15 or 30 minutes in media without 23dMB-PP1 (D).
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 (+/−SD). 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.
Figure 3.
Mps1 kinase activity promotes localization of Mad1, Mad2, Bub1 and Cdc20 to unattached kinetochores.
A, B: Immunolocalization of Mad1, Mad2, Bub1, BubR1, CENP-E, Cdc20, and centromeres (ACA) in HCT-derived (A) or UTR-derived (B) cells treated with nocodazole and MG132 for 1 hour in combination with DMSO or 23dMB-PP1. Graphs represent quantifications of kinetochore signal intensities for the investigated proteins at all kinetochores in a single cell as a ratio of the ACA signal. Data are average of three experiments, 10 cells per experiment (+/− SD). Representative images are shown.
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.
Figure 5.
Mps1 inhibition accelerates mitosis.
A–C: Time-lapse analysis by DIC microscopy of mitotic progression of HCT-derived (A, B) and UTR-derived (C) cells treated with DMSO or 23dMB-PP1. Images in A display morphology of HCT-derived cells imaged at the indicated timepoints after NEB (nuclear envelope breakdown). Graphs in B and C show cumulative percentages of cells that display first signs of furrowing at indicated times after NEB. Dashed lines indicate half-time to furrowing.
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 (+/−SD). 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 (+/−SD). 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.
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.