FIGURE 1:

KIF21B motors stabilize MT dynamics and promote MT assembly. (A) Motor constructs used for single molecule experiments. (B) Kymograph plots showing dynamic MTs polymerizing from stabilized MT seeds in the absence or presence of KIF21B-FL-Halo or KIF21B-MD-Halo motors. Scale bars: 10 µm (horizontal) and 30 s (vertical). (C–E) Average MT plus-end growth distances. Ten kymographs were averaged for each condition. N = 3 independent experiments. (F) MT plus-end growth rates in the presence of increasing concentrations of KIF21B-FL-Halo or KIF21B-MD-Halo motors. Plotted are means and SDs. Kruskal–Wallis one-way ANOVA and Dunn’s multiple comparison (n.s. p > 0.05; *p < 0.05; ***p < 0.001; ****p < 0.0001). Data from 54–94 MTs and N = 3–6 independent experiments. G) MT plus-end catastrophe frequency in the presence of increasing concentrations of KIF21B-FL-Halo or KIF21B-MD-Halo motors. Plotted are means and SDs. Kruskal–Wallis one-way ANOVA and Dunn’s multiple comparison (****p < 0.0001). Data from 54–94 MTs and N = 3–6 independent experiments. (H–J) TIRF microscopy images showing MT formation from free tubulin dimers incubated in the absence or presence of 50 nM KIF21B-FL-Halo or KIF21B-MD-Halo. Images were taken 10 min after solutions were introduced into flow chambers. Scale bar: 10 µm. (K–M) Averaged light scattering traces for solutions containing buffer, Taxol, or 50 nM KIF21B-FL-Halo or KIF21B-MD-Halo motors with 1 mM ATP or 1 mM ADP. Means were compared with Kruskal–Wallis one-way ANOVA and Dunn’s multiple comparison (Buffer–Taxol ****p < 0.0001; Buffer–KIF21B-FL-Halo ****p < 0.0001; KIF21B-FL-Halo ATP–KIF21B-FL-Halo ADP n.s. p > 0.05; Buffer–KIF21B-MD-Halo n.s. p > 0.05; KIF21B-MD-Halo ATP–KIF21B-MD-Halo ADP n.s. p > 0.05). Data from 7–22 traces from N = 5 independent experiments. Graphs show mean values for each time point. See Supplemental Figure S2 for graphs with SDs.