Journal: Biophysical Journal

Article Title: Kinesin-1 autoinhibition tunes cargo transport by motor ensembles

doi: 10.1016/j.bpj.2025.08.032

Figure Lengend Snippet: ( A ) Schematic representation of multimotor motility assay with fluid-like liposome as cargo. Liposome ( gold ) has 5–20 kinesins ( purple ) bound to its surface. The fluorescence of the liposome is tracked by TIRF microscopy as it is moved along MTs ( blue ) by the bound kinesins. ( B ) Dot plots with overlaid violin plots representing multimotor run lengths measured for KinΔC ( cyan ) and K543 ( magenta ) with up to 20 bound kinesins. Black overlaid boxplot shows median and upper and lower quartiles per condition. K543 control data are replotted from Bensel et al., 2024. p -values are as follows: N.S., p > 0.05; * p < 0.05; ** p < 0.01. Run length data are compared using a Kruskal-Wallis with Dunn’s post hoc test using the Benjamin-Yuketieli adjustment. See Table S1 for exact p -values. For KinΔC data, N events ranges from 137 to 480 from at least three independent experiments. ( C ) Dot plot with overlaid violin plot representing multimotor velocities measured for KinΔC ( cyan ) and K543 ( magenta ). Black overlaid boxplot shows median and upper and lower quartiles per condition. K543 control data are replotted from Bensel et al., 2024. p -values are as follows: N.S., p > 0.05; * p < 0.05; ** p < 0.01. Velocity data are compared using a Kruskal-Wallis with Dunn’s post hoc test using the Benjamin-Yuketieli adjustment. See Table S1 for exact p -values. N values are the same as in (B). ( D ) Bar graph depicting the percentage of runs for each condition (single kinesin Qdot, and 5, 10, and 20 kinesins per liposome), which reach the end of the MT before detaching. K543 data are replotted from Bensel et al., 2024. End event frequencies are compared using a two-proportions Z-test. p -values are as follows: N.S., p > 0.05; * p < 0.05; ** p < 0.01. See Table S1 for exact p -values. N values are the same as in ( B ) and ( C ). ( E ) Schematic representation of multimotor optical trapping assay with lipid-coated bead as cargo. Kinesins ( purple ) are bound to lipid-coated beads ( gray ) in a 20 to 1 excess. The laser trap ( red ) is used to position the bead close to an MT ( blue ), and force ramps are recorded. ( F ) Sample force ramp collected for a lipid-coated bead coated with 20 KinΔC motors. Raw data are shown in gray with median-filtered data overlaid in black. ( G ) Detachment force histogram with overlaid double-Gaussian fit collected for KinΔC experimental results ( cyan ). Fit parameters ± fitting errors are shown in inset. A 2 is defined as 1 − A 1 and is not an independently fit parameter with error determined by error propagation. N events = 169 from five independent experiments. ( H ) Detachment force histogram with overlaid triple Gaussian fit collected for K543 ( magenta ). K543 data and fit are replotted from Bensel et al., 2024. Fit parameters ± fitting errors are shown in inset. A 3 is defined as 1 − (A 1 + A 2 ) and is not an independently fit parameter with error determined by error propagation.

Article Snippet: Kinesin-Qdot complex was diluted 1:20 in Motility Buffer (Buffer 1 with 2 mM MgATP, 20 μM paclitaxel, 0.5 mg/mL Casein, 0.5% w/v Pluronic F-127, 5 mM creatine phosphate, 0.4 mg/mL creatine phosphokinase, 10 mM DTT, 3.5 mg/mL glucose, 40 μg/mL glucose oxidase, and 27 μg/mL catalase) and flowed into the motility chamber.

Techniques: Motility Assay, Fluorescence, Microscopy, Control