Fig 1.
Motility properties of kinesin-II at the single-molecule level.
A. Design of a labeled heterodimeric kinesin-II construct. B. Schematic of the in vitro motility assay. Single sfGFP-labeled kinesin-II molecules are observed moving along unlabeled, surface-immobilized microtubules. C. Typical kymograph (space-time plot) of individual sfGFP-kinesin-II motors obtained from single molecule TIRF motility assays. Time is progressing from top to bottom, length from left to right; scale bars: 1 μm (horizontal) and 2 s (vertical); the kinesin-II concentration was 200 pM. D. Mean displacement versus time-lag plot obtained from mean displacements extracted from 237 individual sfGFP-kinesin-II trajectories. Error bars indicate standard error of the mean (SEM). Red: linear fit with slope 0.33 ± 0.01 μm/s (R2 = 0.99). E. Cumulative probability distribution of lengths (μm) of individual trajectories. Red: exponential fit yielding an average run length of 1.18 ± 0.07 μm (R2 = 0.98).
Fig 2.
Kinesin-II motility under crowded conditions.
A. Schematic of the in vitro motility assay. kinesin-II motors walk along the microtubules attached to the glass slide. The fluorescently labeled motor proteins are excited and imaged using TIRF microscopy. B. Kymographs (scale bars: 1 μm (horizontal) and 2 s (vertical)) of kinesin-II motility extracted from time series of TIRF images. The total concentration of labeled plus unlabeled motors is indicated. sfGFP-kinesin-II concentration is 5 nM in all kymographs. Measurements were performed in PEM12 buffer.
Fig 3.
Correlation imaging measurements at high density of kinesin-II.
A. An example of one of the experiments; time series of TIRF images of fluorescently labeled kinesin-II motor proteins on a microtubule at a concentration of 5 nM in PEM12 buffer, exposure time 0.2 s, 1 pixel corresponds to 0.08 μm. The plus and minus ends of the microtubule are indicated. B. Cross sections of the correlation surface at different time lags. Gaussian fits (dashed red line) are used to obtain the peak position and area under these curves. C. Peak position as a function of time. The red linear fit yields the motor velocity of 0.30 ± 0.02 μm/s, R2 = 0.99 D. Area under the curve as a function of time in semi-logarithmic representation. An exponential fit (red) yields the detachment rate of motors, from which the average run length can be determined to be 1.2 ± 0.3 μm, R2 = 0.96.
Fig 4.
Kinesin-II motility parameters as a function of total motor concentration (green), compared to previously published data and simulations from kinesin-1 (blue) and OSM-3 (red) [7]. A. Motor density (total density of labeled and unlabeled motor proteins) on microtubules as function of total motor concentration, calculated from fluorescence intensity fluctuations on ~70 microtubule segments. Light green diamonds: data points of individual microtubule segments (kinesin-II); symbols with error bars: averages and standard deviations calculated at each concentration; curves: predictions of the extended TASEP-LK model. B. Motor velocity as a function of motor density on microtubules. Light green diamonds: velocity determinations on individual microtubule segments (kinesin-II); symbols with error bars: averages and standard deviations of multiple velocity determinations within logarithmically scaled density intervals; curves: predictions of the extended TASEP-LK model. C. Run length as a function of motor density on microtubules. Light green diamonds: run length determinations on individual microtubule segments; symbols with error bars: averages and standard deviations of multiple run length determinations within logarithmically scaled density intervals; curves: predictions of the extended TASEP-LK model.
Table 1.
Fitting and derived parameters for the modified TASEP-LK model for kinesin-II in PEM12 buffer.