Regulation of kinetochore–MT interactions by Hec1 phosphorylation. (A) Schematic of the KMT interface with repetitive MT binding sites (circles), each containing multiple NDC80 complexes. (B) Schematic of the NDC80 complex and location of Aurora B phosphorylation sites in the Hec1 tail. N, N terminus. (C) Fluorescence images of PtK1 cells depleted of endogenous Hec1 and rescued with phosphomimetic versions of Hec1 in an A background fused to GFP. Cells were fixed after a brief incubation in ice-cold media to reduce the number of nonkinetochore spindle MTs. Bar, 10 µm. (D) Quantification of chromosome alignment phenotypes for the Hec1 silence/rescue experiment. At least 108 cells were analyzed for each Hec1 mutant from three separate experiments.