Cdt1 exhibits synergy with the Ska1 and the Ndc80 complexes in binding to microtubules in vitro. (A) Schematics representation of the single-molecule binding assay. (B) Selected images showing the binding of 1 nM GFP-tagged Cdt1^92-546 (indicated as GFP-Cdt1 for all in vitro assays) on the Alexa 647-biotin-labeled microtubules either alone (top) or in the presence of 100 nM untagged SKA1/2 dimer (bottom). Scale bar, 5 μm. (C) Data showing synergic-binding experiment of 1 nM GFP-Cdt1 with varying concentrations of untagged SKA1/2 (10 nM-2 µM), data are mean ± SEM. (D) Same as C but enrichment of 1 nM GFP-SKA1/2 with varying concentrations (10 nM-2 µM) of untagged Cdt1^92-546 (indicated as untagged Cdt1 for all in vitro experiments) was plotted in this case. N ≥ 2 experiments, n ≥ 30 microtubules for each point, data are mean ± SEM. (E) Selected images showing synergic-binding experiment of 10 nM Ska1/2/Ska3-GFP complex with varying concentrations of untagged Cdt1 (0–1 µM). (F) Quantification Ska3-GFP intensity as a function of Cdt1 concentration n ≥ 33 microtubules for each point, data are mean ± SEM. (G) Data showing synergic binding experiment of 1 nM GFP-Cdt1 with varying concentrations of HEC1/NUF2-GFP (10 nM-2 µM), data are mean ± SEM. (H) Selected images showing the binding of HEC1/NUF2-GFP (1 nM) on the Alexa 647-biotin-labeled microtubules either alone (top) or in the presence of 100 nM untagged Cdt1 (bottom). Scale bar, 5 μm. (I) Analysis of data from H showing quantification of synergistic binding between HEC1/NUF2-GFP (1 nM) and varying concentrations untagged Cdt1. N ≥ 2 experiments, n ≥ 30 microtubules for each point, data are mean ± SEM.