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Kinesin expands and stabilizes the GDP-microtubule lattice

Nature Nanotechnology volume 13, pages 386–391 (2018)Cite this article

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Abstract

Kinesin-1 is a nanoscale molecular motor that walks towards the fast-growing (plus) ends of microtubules, hauling molecular cargo to specific reaction sites in cells. Kinesin-driven transport is central to the self-organization of eukaryotic cells and shows great promise as a tool for nano-engineering1. Recent work hints that kinesin may also play a role in modulating the stability of its microtubule track, both in vitro2,3 and in vivo4, but the results are conflicting5,6,7 and the mechanisms are unclear. Here, we report a new dimension to the kinesin–microtubule interaction, whereby strong-binding state (adenosine triphosphate (ATP)-bound and apo) kinesin-1 motor domains inhibit the shrinkage of guanosine diphosphate (GDP) microtubules by up to two orders of magnitude and expand their lattice spacing by ~1.6%. Our data reveal an unexpected mechanism by which the mechanochemical cycles of kinesin and tubulin interlock, and so allow motile kinesins to influence the structure, stability and mechanics of their microtubule track.

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Fig. 1: Strong-binding-state kinesins inhibit GDP-microtubule shrinkage.
Fig. 2: Microtubules are stabilized when kinesins bind to one side of the lattice.
Fig. 3: Subsets of PFs are stabilized by a kinesin-coated surface.
Fig. 4: Nucleotide-free motor domains can bend-lock microtubules.
Fig. 5: Kinesin increases the lattice spacing of GDP microtubules.

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Acknowledgements

The authors thank D. R. Drummond and N. Sheppard for assistance with protein purification, and T. A. McHugh for commenting on the manuscript. This research was funded by the Biotechnology and Biological Sciences Research Council (grant number BB-G530233–1) via the Systems Biology Doctoral Training Centre, University of Warwick; and the Wellcome Trust (grant number 103895/Z/14/Z).

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Authors and Affiliations

  1. Centre for Mechanochemical Cell Biology, Warwick Medical School, Coventry, UK

    Daniel R. Peet & Robert A. Cross

  2. Warwick Systems Biology Centre, University of Warwick, Coventry, UK

    Daniel R. Peet & Nigel J. Burroughs

  3. Mathematics Institute, University of Warwick, Coventry, UK

    Nigel J. Burroughs

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Contributions

D.R.P. and R.A.C. designed the experiments. N.J.B. provided mathematical insight. D.R.P. designed the analyses, collected and analysed the data, developed the microfluidics interface and produced the manuscript and figures. All the authors contributed towards the discussion and interpretation of results, and editing the manuscript.

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Correspondence to Robert A. Cross.

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Supplementary information

Supplementary Information

Supplementary Figures 1–5, Supplementary Tables 1–2, Supplementary methods, Supplementary references.

Supplementary Movie 1 | A kinesin surface-clamp assay

The image data (top) corresponds to the kymograph in Fig. 2c (bottom). A minus-end trail is clearly seen in the no-nucleotide phase. Addition of ADP causes the microtubule tips to shrink. In this case, the minus-end trail is retained during shrinkage. Microtubules are re-stabilized upon addition of taxol and ATP, and the resulting kinesin-driven microtubule gliding reveals the microtubule polarity.

Supplementary Movie 2 | Strong-binding state kinesin can lock the curvature of GDP-microtubules.

For each concentration of T93N, images are sorted according to the microtubule orientation. The marked microtubules in each row (orange asterisks) fall into the orientation range depicted by the protractor diagrams (left). Microtubules are straight and dynamically unstable at the beginning of the movie. Arrows (top) highlight the presence and direction of hydrodynamic flow, which causes microtubule bending. In the absence of kinesin, stopping the flow causes the microtubules to re-straighten and continue to depolymerize. Microtubule curvature is preserved at low concentrations of T93N but not at high concentrations. Microtubules also transiently crinkle when T93N is flowed through at high concentrations.

Supplementary Movie 3 | Kinesin reversibly expands microtubules under constant hydrodynamic flow

The movie corresponds to the microtubule shown in Fig. 5b. As kinesin and ADP are alternately introduced into the flow, the microtubule visibly expands and contracts, most obviously seen by the downstream microtubule tip visibly shifting right and left. Both surface-free and transient surface-snagging behaviour can be seen in this movie, and the microtubule expands to the same extent in each case (Fig. 5d).

Supplementary Movie 4 | Microtubules briefly crinkle when kinesin is introduced to the flow

An ADP-to-kinesin transition is shown, during which the microtubule crinkles and thereby dips in and out of the TIRF illumination. Most of the microtubules are visibly free from the surface for the duration.

Supplementary Movie 5 | Kinesin increases the lattice spacing of surface-stitched GDP-microtubules

The movie corresponds to the microtubule shown in Fig. 5g. Part way through the movie, 200 nM of monomeric kinesin (K340) was flowed through the channel and the microtubule extends and bows so as to follow a longer path length. Flushing with 1 mM ADP triggers kinesin unbinding, and the microtubule reverts to its original length. After washing the sample thoroughly with buffer, the process can be repeated. After the first cycle, the microtubule becomes tethered to the surface at a greater number of interaction sites. During the second kinesin flow-through, part of the microtubule briefly goes out of focus but it is recruited back into the optical plane, demonstrating that our protocol restricts motion in the z-axis to permit reliable quantification. Arrows indicate when the solution is flowing. Scale bar, 10 µm.

Supplementary Animation 1 | Kymograph profile matching for measuring microtubule expansion

The ‘original’ kymograph shown is the same as in Fig. 5b. The ‘transformed’ kymograph is the same image but each row has been compressed by the values shown in Fig. 5d. This highlights the uniform expansion of the microtubule that occurs when kinesin binds.

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Peet, D.R., Burroughs, N.J. & Cross, R.A. Kinesin expands and stabilizes the GDP-microtubule lattice. Nature Nanotech 13, 386–391 (2018). https://doi.org/10.1038/s41565-018-0084-4

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