Transient increase of binding affinity of GDP microtubules by pretreatment with KIF5C. (A) Images of the KIF5C (magenta) motor domain and GDP microtubules (green) in each step of the experiment. First, GDP microtubules were incubated with 10 pM KIF5C in the absence of nucleotide (left column). Following incubation with 100 pM KIF5C (second left column), KIF5C was washed out from the observation chamber using 0.5 M Pipes buffer, and microtubules were incubated in the observation solution (second right column). After the incubation time indicated left side of the images, 10 pM KIF5C was reintroduced in the chamber (right column). (B) Incubation time dependency of the Kd of KIF5C and GDP microtubule after temporal binding of 100 pM KIF5C. Data points are shown in black. Kd values before incubation with 100 pM kinesin and during the incubation with 100 pM kinesin are shown in gray for comparison. The time constant for the decay was estimated to be 2.1 ± 1.1 min (mean ± SEM) by fitting with an exponential decay (gray curve). Nmicrotubules are 35, 43, 28, and 34, and Nbound are 74, 51, 20, and 11 for the data points for 1, 2, 4, and 8 min, respectively.
