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. 2017 Nov;18(11):1947-1956.
doi: 10.15252/embr.201744097. Epub 2017 Sep 8.

Kinesin-2 motors adapt their stepping behavior for processive transport on axonemes and microtubules

Willi L Stepp  1 , Georg Merck  1 , Felix Mueller-Planitz  2 , Zeynep Ökten  3   4
Affiliations

Kinesin-2 motors adapt their stepping behavior for processive transport on axonemes and microtubules

Willi L Stepp et al. EMBO Rep. 2017 Nov.

Abstract

Two structurally distinct filamentous tracks, namely singlet microtubules in the cytoplasm and axonemes in the cilium, serve as railroads for long-range transport processes in vivo In all organisms studied so far, the kinesin-2 family is essential for long-range transport on axonemes. Intriguingly, in higher eukaryotes, kinesin-2 has been adapted to work on microtubules in the cytoplasm as well. Here, we show that heterodimeric kinesin-2 motors distinguish between axonemes and microtubules. Unlike canonical kinesin-1, kinesin-2 takes directional, off-axis steps on microtubules, but it resumes a straight path when walking on the axonemes. The inherent ability of kinesin-2 to side-track on the microtubule lattice restricts the motor to one side of the doublet microtubule in axonemes. The mechanistic features revealed here provide a molecular explanation for the previously observed partitioning of oppositely moving intraflagellar transport trains to the A- and B-tubules of the same doublet microtubule. Our results offer first mechanistic insights into why nature may have co-evolved the heterodimeric kinesin-2 with the ciliary machinery to work on the specialized axonemal surface for two-way traffic.

Keywords: axonemes; heterodimeric kinesin‐2; intraflagellar transport; microtubules.

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Figures

Figure 1
Figure 1. Single‐molecule transport parameters of the full‐length KLP3A/B and KLP11/20 motors on microtubules and axonemes
  1. A–C

    The movement of single KLP3A/B motors fluorescently labeled at the KLP3A subunit was tracked on surface‐attached microtubules at saturating ATP conditions. The velocity (B) and run length data (C) were fit to a Gaussian and single‐exponential distribution, respectively.

  2. D–F

    The corresponding analysis of the KLP11/20 motor fluorescently labeled at the KLP11 subunit moving on surface‐attached axonemes.

Data information: Speeds are fit to a Gaussian distribution (± width of distribution), and run length is fit to a single exponential (± confidence interval).
Figure 2
Figure 2. Impact of the C‐terminal random‐coil domains on the run length of the KLP3A/B motor

  1. Removal of the random‐coil domain of the KLP3A subunit failed to interfere with the transport parameters of the KLP3A/B motor.

  2. In contrast, removing the corresponding domain of the KLP3B subunit reduced the run length by ˜50% without substantially affecting the velocity of the KLP3A/B motor.

  3. Removal of both random‐coil domains had the same effect as the removal of the KLP3B C‐terminal domain (B).

Data information: The P‐values for the statistical tests were obtained from two‐sample t‐tests (A = KLP3AN‐Snap 1‐597/B, B = KLP3AN‐Snap/B1‐592, C = KLP3AN‐Snap 1‐597/B1‐592, FL = KLP3AN‐Snap/B). Speeds are fit to a Gaussian distribution (± width of distribution), and run length is fit to a single exponential (± confidence interval).
Figure 3
Figure 3. Kinesin‐2 motors differentiate between the microtubule and axoneme surface to take processive steps
Tracking of the fluorescently labeled head domains KLP3A and KLP11 of the heterodimeric kinesin‐2 motors at limiting ATP concentrations. Note the corresponding colors in upper and lower panels.
  1. A, B

    The step size distribution centered around ˜13 nm when walking on microtubules. Together with the double‐exponential decay of the dwell times (E, F), these results support a hand‐over‐hand type stepping of the respective motors.

  2. C, D

    Tracking on axonemes increased step size distribution to ˜16 nm which is consistent with protofilament tracking.

  3. E, F

    Double‐exponential decay of the dwell times and raw stepping data with detected steps.

  4. G, H

    Double‐exponential decay of the dwell times again argues for a hand‐over‐hand stepping mechanism on axonemes.

Data information: (E–H) Steps are shown with the detected stepping pattern in red and the calculated step size in nm. Scale bars are 5.04 s (10 frames) wide and 10 nm high. The respective step sizes are fit to a normal distribution (x is mean, and μ is width of the distribution). Dwell times are fit to a double‐exponential distribution (± confidence interval). See Fig EV1 for more step data and Appendix Fig S7 for the respective kymographs. A two‐sample t‐test has confirmed the statistical significance of the difference between the step size distributions on microtubules versus axonemes (P‐values of 3e‐5 for KLP11/20 and 0.05 for KLP3A/B, respectively).
Figure EV1
Figure EV1. Single fluorophore tracking of KLP11/20 and KLP3A/B motors at limiting ATP concentrations
  1. A, B

    Photons from a single fluorophore detected on the CCD are localized and tracked over time. Localization is achieved by fitting a two‐dimensional Gaussian profile to the spot. The Euclidean distance of each location to the position in the first frame is calculated and plotted in blue. Steps in the data are detected by a gliding t‐value algorithm and plotted in red. Frames have a cycle time of 505 ms.

Figure 4
Figure 4. Example trajectories of single KLP11/20 molecules tracked on axonemes (A) and microtubules (B)
  1. A, B

    On the rotationally symmetric microtubule filaments, the motors show a sinusoidal trajectory, corresponding to a periodic helical path around the symmetry axis of the filament. The axoneme is lacking this symmetry due to the combination of the A‐ and B‐tubules into a doublet microtubule along with the interconnection between the respective doublet tubules. Without a symmetry axis, the periodic path is lost and the motor is restricted to a straight path. The dashed lines underlining the respective trajectories have been obtained from a subpixel detection of filament positions (see Materials and Methods for details). Images of the filaments are shown in Appendix Fig S9, and respective kymographs are shown in Fig EV2.

Figure EV2
Figure EV2. Kymographs of KLP11/20 walking on axonemes (A) and microtubules (B)
  1. A, B

    The numbers correspond to the runs shown in Fig 4A and B.

Figure EV3
Figure EV3. Dependence between the trajectory and the step size distribution of a motor taking sidesteps
The stepping of a motor was modeled on a cylinder‐shaped geometry with binding sites distributed as expected for the microtubule lattice 72. A sidestepping probability of 50% was assumed, and 200 steps were simulated. The data were projected onto a plane to simulate a microscope image. For the histogram, step sizes were blurred by a Gaussian distribution of width 3 nm corresponding to an estimated detection accuracy. The pitch for a 13 protofilament microtubule is calculated from the mean step size (x) and the probability for sidesteps (P).
Figure 5
Figure 5. Proposed model for two‐way traffic on a single microtubule doublet
The organization of large intraflagellar trains for a collision‐free bi‐directional transport on a single microtubule doublet within such a spatially restricted environment as seen in vivo poses a remarkable challenge. With the inter‐doublet connections acting as a structural barrier, the motors’ intrinsic ability to switch protofilaments to the left 55, 71 would allow a collision‐free two‐way traffic by separating the large intraflagellar trains to either side of the doublet microtubule. This model may explain why nature has not recycled any other efficient transporter as kinesin‐1 but opted to co‐evolve heterodimeric kinesin‐2 motors with an intrinsic left‐handedness. Whether dynein‐2 displays an exclusive left‐handedness is, however, not yet known.
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References

    1. Block SM, Goldstein LS, Schnapp BJ (1990) Bead movement by single kinesin molecules studied with optical tweezers. Nature 348: 348–352 - PubMed
    1. Mehta AD, Rock RS, Rief M, Spudich JA, Mooseker MS, Cheney RE (1999) Myosin‐V is a processive actin‐based motor. Nature 400: 590–593 - PubMed
    1. Howard J, Hudspeth AJ, Vale RD (1989) Movement of microtubules by single kinesin molecules. Nature 342: 154–158 - PubMed
    1. Svoboda K, Schmidt CF, Schnapp BJ, Block SM (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365: 721–727 - PubMed
    1. Woehlke G, Schliwa M (2000) Walking on two heads: the many talents of kinesin. Nat Rev Mol Cell Biol 1: 50–58 - PubMed

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