The microtubule-binding domain of HEC1 directs MPS1 localization and function. (A) Time-lapse analysis of duration of mitotic arrest in nocodazole- and ZM447439 (ZM)-treated Flp-in HeLa cells transfected with mock or HEC1 siRNA and expressing the indicated GFP-HEC1 proteins. Data indicate cumulative percentages of cells (from a total of ≥125 cells per treatment) that exit mitosis (scored as cell flattening) at the indicated times after NEB and are representative of three independent experiments. (b–d) Representative images (b) and quantification (c and d) of immunolocalization of MPS1, the indicated GFP-HEC1 proteins, and centromeres (CREST) in nocodazole-treated Flp-in HeLa cells transfected with mock or HEC1 siRNA. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in c displays total kinetochore intensities (±SEM) of the indicated proteins relative to centromeres (CREST). Data are from a total of ≥103 cells per treatment from two experiments. Ratios are set to 1 for mock RNAi–treated cells (MPS1) and for GFP-HEC1^WT–expressing cells (GFP-HEC1). Graph in d displays total kinetochore intensities of the indicated proteins relative to centromeres (CREST) for all cells of a single experiment. (e and f) Representative images (e) and quantification (f) of immunolocalization of MPS1, the indicated LacI-GFP-HEC1 proteins, and centromeres (CREST) in nocodazole-treated U2OS-LacO cells. DNA (DAPI) is shown in blue. Insets show magnifications of the boxed regions. Graph in f displays total intensities (±SEM) of MPS1 at LacO arrays relative to LacI-GFP-HEC1 (GFP) and total intensities of LacI-GFP-HEC1. Data are from a total of ≥17 cells from two experiments. Ratios for LacI-GFP-HEC1^WT–expressing cells are set to 1. Bars, 5 µm. WT, wild type; a.u., arbitrary unit.