Fig. 2: The β-hairpin motif mediates kinesin-2 autoinhibition.
From: Regulation of kinesin-2 motility by its β-hairpin motif
a, Composite TIRF images of the 488-nm channel (MT) and 640-nm channel (Alexa-Fluor-647-labeled Kif3A), offset in y axis by 8 pixels (px). Top, with 1 mM ATP. Bottom, with 1 mM AMPPNP. MT; microtubule. b, Left, schematic of the MT gliding assay with biotinylated Kif3AB. Middle, example kymograph. Right, plot of gliding velocities from three technical replicates. Colored circles; individual data points. White circles; average from each separate replicate. Lines, mean ± s.e.m., n = 33, 25 and 28 microtubules analyzed per replicate. c, Left, single-molecule velocity, run length and microtubule landing rate of Alexa-Fluor-647-labeled Kif3AB constructs on microtubules. Constructs were progressively truncated from their C termini, as indicated in the schematics above the plots. Measurements were taken from three technical replicates from separate motility chambers, with three fields of view per replicate. For velocity and run length, the numbers of measurements per technical replicate are: Kif3AΔ89B, n = 120, 100 and 120; Kif3AΔ103B, n = 113, 123 and 106; Kif3AΔ103B Δ39, n = 121, 105 and 102; Kif3AΔ103BΔ90, n = 144, 108 and 103; Kif3AΔ103BΔ114, n = 105, 102 and 116; Kif3AΔ103BΔ156, n = 124, 108 and 104. For landing rate, n = 5 microtubules per replicate. Gray circles, average from each replicate. Lines, mean ± s.e.m. For Kif3A–Kif3B, Kif3AΔC38–Kif3B, and Kif3AΔC59–Kif3B constructs, no motile events were observed across three replicates (depicted with a cross), suggestive of strong autoinhibition at the single-molecule level. For other constructs, velocity and run-length values were not significantly different than each other (P > 0.1 one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test). Landing-rate values for Kif3AΔ103BΔ90, Kif3AΔ103BΔ114 and Kif3AΔ103BΔ156 were significantly higher than those of other constructs (P < 0.05). Exact P values are given in the Methods. Right, an example kymograph of Kif3AΔC103BΔC156. d, Example kymographs of Kif3AB β-hairpin mutants. Kif3AKK mutant: E612K D613K. Kif3BKKK mutant: E607K E608K E609K. Kif3AGly-mut: H609G W610G W617G Q618G. Motility parameters for all constructs are given in Extended Data Table 1.
