Fig. 5: Kinesin-1 pulled out tubulin subunits from the GDP-microtubule lattice.

a Experimental scheme of the motor-pulling assay. GDP-tubulin subunit attached to a DNA linker (3.8 kb) was pulled by biotinylated kinesin-1 molecules along the microtubule axis. The GDP-microtubules were stabilized with GMPCPP-tubulin caps on both ends (not shown for simplicity). b Example kymograph of kinesin-1 stretching a DNA-linker (stained by SYTOX Green) and pulling out a GDP-tubulin subunit. The dissociation of kinesin-1 is indicated with yellow arrowheads. The rupture event corresponding to the dissociation of tubulin was marked by the white arrow. c Time-lapse images of the last stretching event before rupture from the kymograph in b. The DNA molecule of interest is indicated with a red arrowhead. d The number of microtubule-linked DNA molecules remaining after introducing 6 nM of kinesin-1 (either non-biotinylated or biotinylated with ~50% stoichiometry) imaged at 1 frame per minute (error bars: SDs). Biotinylated kinesin-1 led to a significant reduction of fluorescent DNA molecules after 5 min as compared to the non-biotinylated kinesin-1 control (two-tailed Welch t-test p = 0.0003; percentage of DNA remained: 59 ± 4% vs. 94 ± 3%; mean ± SD, n = 3 experiments). e Time-to-rupture (error bars: median ± 95% confidence interval) decreased with increasing concentration of biotinylated kinesin-1 to a value of ~8 s (dashed line). Data collected from triplicate experiments with n = 108, 105, 130, 122, 112, 99 events at each concentration (from 0.5 nM to 9 nM). Rupture time from taxol-stabilized microtubules is shown in blue (n = 160 events from triplicates). f Examples of stepwise increase in fluorescence intensity corresponding to the stepwise increase in force due to the increase in the number of motors engaged in force generation (Supplementary Movie 2 corresponding to the kymograph on the top). An additional example is included in Supplementary Figure 5b,c.