Phosphoregulation of the KMT interface in prometaphase cells. (A) Mean KMT half-life calculated with the two models relative to these values measured previously in PtK1 cells in prometaphase and metaphase (3.5 and 9 min, respectively; Cimini et al., 2006; DeLuca et al., 2006). Both models were calibrated such that the interfaces with 1D NDC80 complexes matched the metaphase half-life. The repetitive sites model predicts very unstable KMT attachments for 2D, 3D, and 4D proteins, so these bars are virtually invisible, whereas the large half-life for noD NDC80 kinetochores is plotted with an interrupted axis. (B) Schematics and representative still images from time-lapse videos of PtK1 cells subjected to a monastrol washout assay. Numbers are times (in minutes) from monastrol washout. Hec1-GFP is shown in green (kinetochores); mCherry-tubulin is shown in red (MTs); cartoon chromosomes are shown in blue; asterisks show position of poles. Bar, 5 µm. (C) Rate of pole separation after monastrol washout. ZM, Aurora B inhibitor ZM447439. The bar labeled Hec1 K166D corresponds to a non-MT–binding Hec1K166D mutant (Ciferri et al., 2008; Sundin et al., 2011). n = 10 cells were analyzed for each Hec1 mutant. (D) Correlation plot for in silico and in vivo data from A and C, respectively. Pearson correlation analysis shows that the correlation is significant for the lawn model but not the repetitive sites model (R² is 0.78 and 0.51, respectively, with threshold value of 0.05). Error bars are SEMs.