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Single-molecule observations of neck linker conformational changes in the kinesin motor protein

Nature Structural & Molecular Biology volume 13, pages 887894 (2006) | Download Citation

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Abstract

Kinesin-1 is a dimeric motor protein that moves cargo processively along microtubules. Kinesin motility has been proposed to be driven by the coordinated forward extension of the neck linker (a 12-residue peptide) in one motor domain and the rearward positioning of the neck linker in the partner motor domain. To test this model, we have introduced fluorescent dyes selectively into one subunit of the kinesin dimer and performed 'half-molecule' fluorescence resonance energy transfer to measure conformational changes of the neck linker. We show that when kinesin binds with both heads to the microtubule, the neck linkers in the rear and forward heads extend forward and backward, respectively. During ATP-driven motility, the neck linkers switch between these conformational states. These results support the notion that neck linker movements accompany the 'hand-over-hand' motion of the two motor domains.

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  • 07 November 2006

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Acknowledgements

We thank K. Thorn for development of the initial version of the microscope system and for discussions, and U. Wiedemann for support in cloning and protein purification. M.T. is supported by grants from the Mitsubishi Foundation, Asahi Glass Foundation, Sumitomo Foundation and Inamori Foundation and by Grants-in-Aid for Scientific Research on Priority Areas. R.D.V. is supported by grants from the Howard Hughes Medical Institute and the US National Institutes of Health.

Author information

Affiliations

  1. Department of Applied Physics, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

    • Michio Tomishige
  2. Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 600 16th Street, San Francisco, California 94158, USA.

    • Michio Tomishige
    • , Nico Stuurman
    •  & Ronald D Vale

Authors

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Contributions

M.T. and R.D.V. conceived and designed the experiments. M.T. performed the experiments and data analysis. N.S. contributed to microscope construction and programming. R.D.V. and M.T. discussed the results and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Michio Tomishige or Ronald D Vale.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Effects of dye-labeling on processivity.

  2. 2.

    Supplementary Fig. 2

    Histograms for monomer FRET.

  3. 3.

    Supplementary Fig. 3

    Dynamic FRET traces for neck linker.

  4. 4.

    Supplementary Fig. 4

    Histograms of dwell time between transitions.

  5. 5.

    Supplementary Fig. 5

    Dynamic FRET traces for controls.

Videos

  1. 1.

    Supplementary Video 1

    Photobleaching events of 342:342 heterodimer kinesin molecules labeled with Cy3 (donor) and Cy5 (acceptor) attached to an axoneme in the presence of 1 M ATP (excited at 514 nm). The sequence shows a molecule that initially has high FRET, and then suddenly the acceptor dye disappears with an accompanying single step recovery of donor fluorescence. This is a clear indication for single dye pair FRET. The donor fluorescence subsequently disappears in a single step, presumably due to photobleaching or detachment from the microtubule. Later, another dual labeled molecule binds to the axoneme and shows similar photobleaching process. Left half and right half of the images show donor and acceptor channels, respectively, which are projected side-by-side on the ICCD camera simultaneously. 1/2 real time. Scale bar, 2 µm.

  2. 2.

    Supplementary Video 2

    Single molecule FRET observation of 215-342 heterodimer kinesin moving along an axoneme in the presence of saturating ATP (1 mM). This video shows three fluorescence spots showing FRET moving processively along an axoneme. The axoneme lies approximately vertical in the image with its plus end pointing upwards. No apparent anticorrelated donor and acceptor intensity change is observed. 1/2 real time. Scale bar, 2 µm.

  3. 3.

    Supplementary Video 3

    Single molecule FRET observations of 215-342 heterodimer kinesin moving slowly along axonemes in the presence of low ATP (1 M). Video 3 and 4 represent the panels shown in Figure 3a, (far left and far right, respectively). Both of the spots move slowly upwards. Anti-correlated donor and acceptor intensity changes are observed. 1/2 real time. Scale bar, 2 µm.

  4. 4.

    Supplementary Video 4

    Single molecule FRET observations of 215-342 heterodimer kinesin moving slowly along axonemes in the presence of low ATP (1 M). Video 3 and 4 represent the panels shown in Figure 3a, (far left and far right, respectively). Both of the spots move slowly upwards. Anti-correlated donor and acceptor intensity changes are observed. 1/2 real time. Scale bar, 2 µm.

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DOI

https://doi.org/10.1038/nsmb1151

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