KKT4 binds and diffuses on microtubules. (A) Wild-type, fluorescent-tagged KKT4 particles (green) decorating a Taxol-stabilized microtubule (magenta). (B) Two-color fluorescence image (left) and corresponding kymograph (right) showing diffusion of the KKT4 particles on the microtubule lattice. (C) Distribution of residence times on microtubules for wild-type KKT4 particles. Lower dotted line shows exponential fit used to determine average residence time (n = 452 binding events on 48 microtubules). Upper dotted line shows exponential distribution of bleach times for single fluorescent-tagged KKT4 particles, corresponding to an average of τ_bleach = 25 ± 1 s (n = 732 bleach events), which is long enough to ensure that KKT4 particles usually detached before bleaching. (D) Mean-squared displacement (MSD) of wild-type KKT4 particles plotted against time. Dotted line shows linear fit used to determine diffusion coefficient (n = 452 events). (E) Distribution of initial brightness values for wild-type KKT4 particles diffusing on Taxol-stabilized microtubules. Data are fitted by the sum of two Gaussians (dashed black curves) corresponding to a large population (72%) with a unitary brightness of 3,140 ± 1,470 a.u. and a small population (28%) with twice the brightness (mean ± SD; n = 452 particles).