Biased use of a subset of GDP microtubules in vitro. (A–F) GDP microtubules (GDP-MT; A–C) or GMPCPP microtubules (GMPCPP-MT; D–F) were anchored by sparsely (three or four sites per micrometer microtubule protofilament) labeled biotin to the avidin-coated glass surface and the dimeric KIF5C moved along the microtubules in the presence of 1 mM ATP. (B and E) Kymographs of KIF5C movement along the microtubules in the presence of 0.12 nM KIF5C. Each kymograph corresponds to the KIF5C movement along the microtubules shown in A and D. Note that brightness of the kymographs is inversed. See also Video 1. (C and F) Time courses of the binding frequency of KIF5C to each microtubule in A and D. (G–I) Histograms of mean KIF5C binding frequency onto GDP (top) and GMPCPP microtubules (bottom) during 100-s incubation with 0.12 nM (G), 1.2 nM (H), and 12 nM (J) KIF5C. GDP microtubules showed lower binding frequency than GMPCPP microtubules under 0.12 nM KIF5C (G; P < 0.00001, Steel–Dwass test). Two-peak distribution with a sharp peak near zero and a broad peak around 0.1 molecule s⁻¹ µm⁻¹ nM⁻¹ was evident with GDP microtubules at lower KIF5C concentrations (G and H; see Tables S1, S2, and S3 for statistical details). The peak around zero disappeared with 12 nM KIF5C (I), and the binding frequency distribution was similar in GDP microtubules and GMPCPP microtubules (I; P = 0.53, Steel–Dwass test). It should be noted that these three KIF5C conditions were examined with the same experimental chambers for GDP microtubules and GMPCPP microtubules, respectively. Thus, the zero peak in GDP microtubules with lower KIF5C concentrations would not be due to collapse of the GDP microtubules. (J–L) Histograms of the off rates (J), velocities (K), and run lengths (L) of the KIF5C dimer on these microtubules. There were no significant differences in these parameters, as summarized in Tables S1, S2, and S3.