Supplementary Figure 4: Properties of dynein teams and comparison between human and yeast dyneins.

(a) Kymograph showing the movement of QDs driven by multiple molecules of full-length human dynein in the presence of 1 mM ATP. (b) Velocity distributions of the moving portion selected from the traces of QD–dynein movements. The velocity was derived from linear-fitting of each trace (1 mM ATP). The directed movements of QD–dynein were selected by visual inspection, unlike the case of Fig. 4c. The movement that moved for over 200 nm was measured. The velocity toward the direction of dynein displacement (MT minus end) is shown as a positive value. The experiments were repeated three times. (c) Kymographs depicting the movement of 2-, 4- and 8-dynein teams in the presence of 1 mM ATP. Figure 4b shows all traces analysed for each construct. (d) Velocity distribution of the moving portion selected from the movement of two coupled full-length dynein molecules. The velocity was derived from linear-fitting of each trace (1 mM ATP, n = 471 from three independent experiments). The directed movements were selected by visual inspection, unlike the case of Fig. 4c. The processive segment that moved over 200 nm was analysed. This histogram can be compared with the histograms in b. The velocity toward the direction of dynein displacement (MT minus end) is shown as a positive value. (e) Comparison of the segmental velocities among different dynein complexes measured at a time interval of 1.0 s. The vertical axis is expressed as density so that the shape of the three distributions can be directly compared. The velocity toward the direction of dynein displacement (MT minus end) is shown as a positive value. The number of traces is 330, 375, 128 and 127 for single, 2-, 4- and 8-dynein teams, respectively. The experiment with single dyneins and other constructs was repeated four and three times, respectively. (f) MSD analysis of the movement of different dynein teams in the presence of 1 mM ATP. The traces of single dynein molecules used in this figure are identical to those in Supplementary Fig. 1d. The traces of 2-, 4- and 8-dynein teams are identical to those in e. (g) Comparison of MSD between single and two coupled dynein molecules in the presence of 1 mM ATP. The traces used are identical to those in f. Each plot represents mean ± s.e.m. The MSD plots were fitted by MSD(t) = v_drift²t² + 2Dt + δ(v_drift, a drift velocity; D, a diffusion coefficient; δ, a constant). The diffusive nature in the inhibited state would allow dynein to explore the MT in search of cargo with low ATP consumption. The thermally driven 1D diffusion covers shorter distances (<0.5 μm) more rapidly than directed movement². (h) More examples of typical forces generated by multiple full-length dynein molecules. The grey line shows the raw trace acquired at 11 kHz. The black line represents the 25-Hz median filtered trace. (i) Cluster mode analysis of the force distribution of multiple full-length dynein (grey bar, the histogram is identical to that shown in Fig. 4f). Each Gaussian distribution (solid line) represents the predicted Gaussian mode. In this case, the best model according to the algorithm was an unequal variance model with 4 clusters. The algorithm provides parameter estimation without any prior knowledge about the number of clusters. The values at the top of Gaussian distributions denote the means of them. (j) Comparison of duration in the strongly bound state (ADP or apo state) between human and yeast monomeric dyneins at no load. The cumulative plots of durations were fitted by a one-phase exponential decay model. The decay constants (± s.e.m. of fitting) are shown. The experiment was conducted three times each.