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Direct observation of intermediate states during the stepping motion of kinesin-1

Nature Chemical Biology volume 12, pages 290–297 (2016)Cite this article

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Abstract

The dimeric motor protein kinesin-1 walks along microtubules by alternatingly hydrolyzing ATP and moving two motor domains ('heads'). Nanometer-precision single-molecule studies demonstrated that kinesin takes regular 8-nm steps upon hydrolysis of each ATP; however, the intermediate states between steps have not been directly visualized. Here, we employed high-temporal resolution dark-field microscopy to directly visualize the binding and unbinding of kinesin heads to or from microtubules during processive movement. Our observations revealed that upon unbinding from microtubules, the labeled heads were displaced rightward and underwent tethered diffusive movement. Structural and kinetic analyses of wild-type and mutant kinesins with altered neck linker lengths provided evidence that rebinding of the unbound head to the rear-binding site is prohibited by a tension increase in the neck linker and that ATP hydrolysis by the leading head is suppressed when both heads are bound to the microtubule, thereby explaining how the two heads coordinate to move in a hand-over-hand manner.

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Figure 1: Structural and kinetic intermediates within the 8-nm forward step of the hand-over-hand movement of kinesin-1.
Figure 2: Observation of the two dimensional motion of the gold probe attached to a kinesin head at 10 μM ATP.
Figure 3: Observation of the biased tethered diffusional motion of the unbound head of kinesin-1.
Figure 4: Observation of the head motion during processive motility under various ATP conditions.
Figure 5: Observation of the head motion for the kinesin mutant with extended neck linker.
Figure 6: Observation of the head motion for the kinesin mutant with truncated neck linker.

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Acknowledgements

We thank M. Nakajima for support with cloning, M. Tanigawara for support with the preparation of colloidal gold and R. Vale for useful comments on the manuscript. We also acknowledge that the neck-linker mutants were first established by M.T. with R. Vale. H.I. is supported by Research Fellowships for Young Scientists from the Japan Society for the Promotion of Science. M.T. (no. 24370063), R.I. (no. 24370062, no. 26104507) and H.N. (no. 25251016) are supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.

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Author notes

  1. Hiroshi Isojima and Ryota Iino: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Applied Physics, School of Engineering, The University of Tokyo, Tokyo, Japan

    Hiroshi Isojima, Yamato Niitani & Michio Tomishige

  2. Okazaki Institute for Integrative Bioscience, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi, Japan

    Ryota Iino

  3. Department of Functional Molecular Science, School of Physical Sciences, The Graduate University for Advanced Studies (SOKENDAI), Kanagawa, Japan

    Ryota Iino

  4. Quantum-Phase Electronics Center, School of Engineering, The University of Tokyo, Tokyo, Japan

    Yamato Niitani

  5. Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo, Japan

    Hiroyuki Noji

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Contributions

M.T. and H.I. conceived and designed the experiments; R.I. constructed and refined the microscope with H.N.; H.I. performed the experiments with R.I.; Y.N. performed the experiments added in the revision; H.I., R.I. and Y.N. analyzed the data and prepared the figures; and M.T. wrote and R.I. edited the manuscript.

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Correspondence to Michio Tomishige.

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Isojima, H., Iino, R., Niitani, Y. et al. Direct observation of intermediate states during the stepping motion of kinesin-1. Nat Chem Biol 12, 290–297 (2016). https://doi.org/10.1038/nchembio.2028

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