Comparison of relative affinity of XCTK2 and Eg5 and XCTK2 single molecule characterization of XCTK2. (A) Comparative quantification of the binding of GFP-XCTK2 and Eg5-GFP to single microtubules (MTs) and microtubule pairs (made with 0.5 nM unlabeled XCTK2) at identical conditions. (left) TIRF microscopy images of Cy5-microtubules (top) and of GFP-labeled motors as indicated (bottom) measured at identical microscopy settings for the two motors. (right) Averaged fluorescence intensities of the two GFP-labeled motors in microtubule overlaps and on single microtubules. A.U., arbitrary unit. (B) Single molecule TIRF microscopy of 0.02 nM GFP-XCTK2 on immobilized microtubules. (left) Kymographs showing binding events on an individual microtubule over time. (middle) Histogram showing the distribution of GFP-XCTK2 dwell times on microtubules and monoexponential fits (red line). The mean dwell time derived from this fit is τ = 2.86 ± 0.08 s. (right) MSD curve generated from 2,186 GFP-XCTK2 binding events and a linear fit (red line; R² = 0.999). The initial linear increase of the MSD curve shows that XCTK2 does not move in a directional manner but diffuses via its nonmotor binding site on the microtubule. The one-dimensional diffusion coefficient D as derived from the slope of the fit (MSD = v²t² = 2Dt) is D = 0.112 ± 0.001 µm²/s. (A and B) Stated errors and error bars are standard errors of the mean. All experiments shown were performed in assay buffer containing 50 mM KCl. Bars: (A and B [horizontal]) 5 µm; (B, vertical) 5 s.