Figure 4. Charge-reversal mutant KKT4 binds more weakly. (A) Kymographs showing binding and diffusion of full-length, wild-type KKT4 and charge-reversal mutant KKT4 (mut) on Taxol-stabilized microtubules. (B) Distributions of residence times on microtubules for wild-type KKT4 (red) and charge-reversal mutant KKT4 (blue). Corresponding dotted lines show exponential fits used to determine average residence times (n > 452 binding events on >48 microtubules). Upper dotted lines show exponential bleach-time distributions for single wild-type and mutant KKT4 particles, corresponding to average bleach times of τbleach = 25 ± 1 s and τbleach = 30 ± 1 s, respectively. Wild-type data are recopied from Fig. 2 for comparison. (C) Mean-squared displacement (MSD) of wild-type KKT4 (red) and mutant KKT4 (blue) particles plotted against time. Dotted lines show linear fits used to determine diffusion coefficients (n > 452 particles). Wild-type data are recopied from Fig. 2 for comparison. (D) Schematic of laser trap assay used to measure friction coefficients and rupture strengths for KKT4115–343-decorated beads. (E) Example record showing trap force and bead displacement versus time. (F) Friction coefficients for wild-type KKT4115–343 (red) and charge-reversal mutant KKT4115–343 (blue) at indicated concentrations (mean ± SEM; n = 13–47 events). All individual friction coefficient values are given in Table S1. (G) Attachment survival probability versus force for wild-type KKT4115–343 (red) and charge-reversal mutant KKT4115–343 (blue; n = 33 and 35 events, respectively). All individual rupture force values are given in Table S1.
Figure 4.

Charge-reversal mutant KKT4 binds more weakly. (A) Kymographs showing binding and diffusion of full-length, wild-type KKT4 and charge-reversal mutant KKT4 (mut) on Taxol-stabilized microtubules. (B) Distributions of residence times on microtubules for wild-type KKT4 (red) and charge-reversal mutant KKT4 (blue). Corresponding dotted lines show exponential fits used to determine average residence times (n > 452 binding events on >48 microtubules). Upper dotted lines show exponential bleach-time distributions for single wild-type and mutant KKT4 particles, corresponding to average bleach times of τbleach = 25 ± 1 s and τbleach = 30 ± 1 s, respectively. Wild-type data are recopied from Fig. 2 for comparison. (C) Mean-squared displacement (MSD) of wild-type KKT4 (red) and mutant KKT4 (blue) particles plotted against time. Dotted lines show linear fits used to determine diffusion coefficients (n > 452 particles). Wild-type data are recopied from Fig. 2 for comparison. (D) Schematic of laser trap assay used to measure friction coefficients and rupture strengths for KKT4115–343-decorated beads. (E) Example record showing trap force and bead displacement versus time. (F) Friction coefficients for wild-type KKT4115–343 (red) and charge-reversal mutant KKT4115–343 (blue) at indicated concentrations (mean ± SEM; n = 13–47 events). All individual friction coefficient values are given in Table S1. (G) Attachment survival probability versus force for wild-type KKT4115–343 (red) and charge-reversal mutant KKT4115–343 (blue; n = 33 and 35 events, respectively). All individual rupture force values are given in Table S1.

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