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. 2013 Apr 15;201(2):217–231. doi: 10.1083/jcb.201210033

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© 2013 Nijenhuis et al.

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Figure 6. — Aurora B regulates MPS1 kinetochore localization by controlling function of the TPR domain. (a and b) Representative images (a) and quantification (b) of immunolocalization of LAP-MPS1^1–192 and centromeres (CENP-A) in prophase HeLaK FRT TetR cells depleted of MPS1 and treated with ZM447439, as indicated. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in b shows total kinetochore intensities (±SEM) of MPS1 relative to centromeres. Data are from ≥38 cells from two experiments. Ratios for mock-treated cells are set to 1. (c and d) Representative images (c) and quantification (d) of immunolocalization of the indicated LAP-MPS1 proteins and centromeres (CENP-A) in MPS1-depleted HeLaK FRT TetR cells treated with nocodazole and reversine, with or without ZM447439. DNA (DAPI) is shown in blue. Insets are magnifications of the boxed regions. Graph in d shows total kinetochore intensities (±SEM) of MPS1 relative to centromeres in DMSO-treated (gray bars) or ZM447439-treated (blue bars) cells. Data are from ≥32 cells from two experiments. Ratios for mock-treated, LAP-MPS1^WT–expressing cells are set to 1. (e) Time-lapse analysis of the duration of mitotic arrest in HeLaK FRT TetR cells transfected with mock or MPS1 siRNA and expressing the indicated LAP-MPS1 proteins and treated with nocodazole and DMSO (top) or nocodazole and ZM447439 (ZM; bottom). Data indicate cumulative percentage of cells (from a total of ≥70 cells) that exit mitosis (scored as chromosomal decondensation) at the indicated times after NEB and are representative of at least two independent experiments. (F) Model of regulated MPS1 localization at unattached kinetochores. See Discussion for details. Bars, 5 µm. WT, wild type.