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. 2022 Aug 2;119(32):e2113795119. doi: 10.1073/pnas.2113795119

Fig. 3.

Fig. 3.

Single-molecule behavior of disease-associated KIF1A mutants. (A) Schematic drawing of the domain organization of KIF1A motor protein and recombinant protein analyzed in Fig. 3. (B) Purified KIF1A(1–393)::LZ::mScarlet and its mutants were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detected by trichloroethanol staining. M represents a marker lane. Numbers on the left indicate molecular weight (kDa). Arrow indicates KIF1A(1–393)::LZ::mScarlet. (C–G) Representative kymographs showing the motility of 10 pM KIF1A(wt) (C), 100 pM KIF1A(R11Q) (D), 10 pM KIF1A(R254Q) (E), 100 pM KIF1A(P305L), and 100 pM KIF1A(wt) (G). Vertical and horizontal bars represent 5 s and 5 μm, respectively. (H) Dot plots showing the velocity of KIF1A. Each dot indicates one datum. Green bars represent mean ± SD. Kruskal–Wallis test followed by Dunn’s multiple-comparison test. n = 433 (wt), 325 (R254Q), and 498 (P305L). ****, Adjusted P value of <0.0001. Note that no processive movement was detected for KIF1A(R11Q). ND, not detected. (I) Dot plots showing the landing rate of KIF1A. The number of KIF1A that bound to microtubules was counted and adjusted by the time window and microtubule length. Each dot shows one datum. Green bars represent median value. Kruskal–Wallis test followed by Dunn’s multiple comparison test. n = 30 (10 pM wt), 28 (100 pM R11Q), 29 (10 pM R254Q), and 30 (100 pM P305L) movies. ****, Adjusted P value of <0.0001. Compared with KIF1A(wt). Note that no landing event was detected in 10 pM KIF1A(R11Q) and KIF1A(P305L) experiments. (J) Dot plots showing the run length of KIF1A. Each dot shows one datum. Green bars represent median value and interquartile range. Kruskal–Wallis test followed by Dunn’s multiple comparison test. n = 312 (wt), 241 (R254Q), and 243 (P305L) homodimers. ****, Adjusted P value of <0.0001. Note that all KIF1A motility events were included, including those that end when the motor reaches the end of a microtubule; thus, the reported run lengths are an underestimation of the motor’s processivity. See also SI Appendix, Fig. S3.