Skip to main content
. 2022 May 12;33(6):ar52. doi: 10.1091/mbc.E21-10-0480

FIGURE 2:

FIGURE 2:

Motor multimerization and KIF21B MTRs improve motor processivity and track switching. (A) Schematic of in vitro single molecule assays. Dynamic MTs were grown from surface-immobilized GMPCPP MT seeds. Some MTs remain isolated in space as they grow, while others overlap in parallel or antiparallel orientations with neighboring MTs. Kymographs show growing dynamic MT ends over time for each type of MT arrangement. Dynamic MT plus ends are indicated (+). KIF21B dimers or multimeric complexes contact MT lattices with their N-terminal motor domains, as well as MTRs within their C-terminal tails. Motor domains were labeled with TMR Halo ligands (cyan dot). Scale bars: 5 µm (horizontal) and 1 min (vertical). (B–D) Kymograph plots of KIF21B-FL-Halo, KIF21B-MD-Halo, and KIF21B-FL-Halo motility in low-ionic-strength buffering system along single MTs or parallel and antiparallel MT bundles. Zoom-in image highlights track switches observed on antiparallel MTs (indicated with white * in expanded images). Track switches were defined as a reversal in run direction exceeding three pixels. Scale bars: 5 µm (horizontal) and 20 s (vertical). Zoom-in scale bars: 2.5 µm (horizontal) and 5 s (vertical). (E–G) Cumulative distribution of total distance motor traveled along different MT arrays. Data points were fitted by a single exponential decay function. Differences in run distances can be more clearly seen in insets. Average distance traveled, as calculated by taking the inverse of the rate constant, and SDs are shown in the legends. Kruskal–Wallis one-way ANOVA and Dunn’s multiple comparison (n.s. p > 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). (H, I) Frequency of track switches for single KIF21B-FL-Halo and -MD-Halo motor runs or single and multimeric KIF21B-FL-Halo motor run along antiparallel MTs. Two-tailed Mann–Whitney (***p < 0.001; ****p < 0.0001). Data for single molecule KIF21B-FL-Halo and KIF21B-MD-Halo from 400–1069 motor runs from n = 20–60 MTs and N = 6–7 independent experiments. Data for single and multimeric KIF21B-FL-Halo from 54–174 motor runs from n = 11–12 MTs and N = 4 independent experiments.