The Ndc80-Cdt1-Ska1 tripartite complex exhibits potent tracking of polymerizing and depolymerizing microtubule plus ends. (A) Schematic representation of a single-molecule dynamic microtubule-binding assay. Arrow shows the direction of microtubule dynamics. (B) Representative time lapse images recorded with surface immobilized microtubules after the addition of a mixture of GFP-Cdt1 (1 nM), untagged SKA1/2 (1 nM), soluble tubulin (1 mg/ml), and Mg-GTP (1 mM), a complex of GFP-Cdt1 and SKA1/2 which lands near the plus-ends is indicated using arrowheads within the boxed areas. Time in s, and the scale bar is 5 µm. (C) A kymograph of the tip-tracking complex in B. (D) Same as in B but in the presence of a mixture of whole Ndc80 complex and Cdt1 (both untagged) with GFP-tagged hexameric SKA1/2/3 complex (1 nM each). Arrowheads indicate the dynamic plus-ends. (E) A kymograph of the tip tracking complex in D. (F) Same as in B but in the presence of a mixture of whole Ndc80 complex and dimeric SKA1/2 (both untagged) with GFP-tagged Cdt1 (1 nM each). Arrowheads indicate the dynamic plus-ends. (G) A kymograph of the tip tracking complex in F. (H) Kaplan-Meier survival curves for the total residence time (s) of tip-tracking complexes formed in the presence of indicated conditions. Calculated from n > 30 events and N > 3 independent trials in each case. (I) Representative time lapse images recorded with surface immobilized microtubules after addition of a mixture of GFP-Cdt1, SKA1/2, Ndc80^{alexa 647} (1 nM each), and soluble tubulin (1 mg/ml), supplemented with Mg-GTP (1 mM). Arrow indicating the direction of movement of the tip-tracking complex. Time is in s, and the scale bar is 5 µm. (J) Kymographs of the tip-tracking complex in GFP-Cdt1 channel and in the Ndc80^{alexa 647} channel. Arrow indicating regrowth event.