FIGURE 3:

Quantitative TIRF analysis of single NDC80 molecules on MTs. (A) Representative kymographs of NDC80 phosphomimetic mutants on MTs. Exposure time: 10 ms. Soluble protein concentration was 50 pM for all proteins. (B) Top row, histograms of the integrated intensities for NDC80-GFP dots on the coverslip-attached MTs, >300 dots for each mutant. Background was subtracted, and data were normalized to the number of GFP molecules. For all proteins, >95% of dots had the intensity of a single GFP (95% confidence); therefore our data reported in Figures 2 and 3 correspond to single NDC80 complexes. Red lines are Gaussian fits. Bottom row, histograms of residency times for individual NDC80 complexes (at least 2000 dots for each protein). Dissociation rates (k_off) for different NDC80 mutants were determined from the exponential fits. (C) MSD vs. time; for each mutant protein the tracks from at least 600 individual NDC80 complexes were averaged; data based on at least two independent experiments for each protein. Symbols are experimental data; lines are linear fits; error bars are SEM. (D) Single-molecule parameters for NDC80 phosphomimetic mutants plotted on a logarithmic scale. Values for noD protein were taken as 100% (Table 1). For 1D and 4D proteins average values are plotted. Bars are mean ± SEM for number of independent experiments (N ≥ 2), in which n > 600 molecules were analyzed for each protein.