(A) Kymograph from experiments with dynamic microtubules illustrating increased velocity of minus end-directed dynein-dynactin-BICD2N complexes in the presence of LIS1. Arrowheads: examples of minus end-directed complexes. Y-axis, time; x-axis, distance. Scale bars, 3 s and 1 μm. (B–C) Quantification of the velocity (B) and run length (C) of minus end-directed dynein-dynactin-BICD2N complexes on dynamic microtubules in the presence and absence of LIS1. See Figure 6—figure supplement 1 for examples of velocity distributions. (D) Quantification of minus end-directed velocity of dynein-dynactin-BICD2N complexes on taxol/GMPCPP-stabilised microtubules in the presence and absence of LIS1. (E) Quantification of fluorescence intensity of Alexa647-dynein in minus end-directed complexes in the presence and absence of LIS1. The experiments were performed with stabilised microtubules. (F) Quantification of LIS1’s inhibitory effect on microtubule gliding by surface immobilised human dynein. Dynein alone or with a 20x or 200x molar excess of LIS1 dimers was mixed and incubated with glass surfaces, followed by washing and addition of fluorescent microtubules. In B–D, means ± S.D. are shown with values for each chamber represented as magenta circles (four chambers per condition in B and C, three chambers per condition in D); mean number of complexes analysed per chamber: 60 (B, C) and 106 (D). In E, means ± S.D. are shown with values for each movie represented as magenta circles (six movies from two chambers per condition, with a mean of 102 complexes analysed per chamber). In F, means ± S.D. are shown with values for each microtubule represented as magenta circles (30 microtubules from two chambers per condition). Statistical significance was evaluated with a one-way ANOVA with Sidak’s multiple comparisons test (B–D and F) or a Mann-Whitney test (E). (****p<0.0001; *** p<0.001; **p<0.01; *p<0.05; ns, not significant). Dynactin complexes, BICD2N dimers and LIS1 dimers were used, respectively, at a molar excess of 2x, 10x and 20x compared to dynein, except in some experiments in D and F, when LIS1 dimers were included at a molar excess of 200x relative to dynein. Dynein concentration in the assembly mixes was 20 nM (with a 1 in 10 dilution added to the imaging chambers) (A–E) or 30 nM (F). The acquisition rate was 7.3 frames/s in A and D, one frame/s in F, and 1.7 frame/s in other panels.