Abstract
The microtubule cytoskeleton is a dynamic structure in which the lengths of the microtubules are tightly regulated. One regulatory mechanism is the depolymerization of microtubules by motor proteins in the kinesin-13 family1. These proteins are crucial for the control of microtubule length in cell division2,3,4, neuronal development5 and interphase microtubule dynamics6,7. The mechanism by which kinesin-13 proteins depolymerize microtubules is poorly understood. A central question is how these proteins target to microtubule ends at rates exceeding those of standard enzyme–substrate kinetics8. To address this question we developed a single-molecule microscopy assay for MCAK, the founding member of the kinesin-13 family9. Here we show that MCAK moves along the microtubule lattice in a one-dimensional (1D) random walk. MCAK–microtubule interactions were transient: the average MCAK molecule diffused for 0.83 s with a diffusion coefficient of 0.38 µm2 s-1. Although the catalytic depolymerization by MCAK requires the hydrolysis of ATP, we found that the diffusion did not. The transient transition from three-dimensional diffusion to 1D diffusion corresponds to a “reduction in dimensionality”10 that has been proposed as the search strategy by which DNA enzymes find specific binding sites11. We show that MCAK uses this strategy to target to both microtubule ends more rapidly than direct binding from solution.
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Acknowledgements
We thank R. Hartmann for cloning and protein purification work; V. Varga for purification of unlabelled MCAK; E. Schäffer for the application of F-127; F. Ruhnow for the two-dimensional gaussian peak-fitting tool; A. Hyman, F. Jülicher, E. Schäffer, I. Riedel, J. Stear, G. Klein, V. Varga, A. Hunt and H. T. Schek for review of the manuscript; and S. Wolfson for proofreading and editing. Andor Technologies lent us the Andor iXon camera, and the VisiTIRF condenser was developed in collaboration with Visitron Imaging Systems, GmbH. This research was supported by the Max Planck Gesellschaft and the NIH.
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Supplementary Notes
This file contains Supplementary Notes, Supplementary Methods and legends for Supplementary Videos. (PDF 1504 kb)
Supplementary Video 1
This movie shows MCAK-induced depolymerization of microtubules. Epifluorescence images (TRITC) of immobilized microtubules were recorded with 10s intervals for 12 minutes. At 4 minutes, buffer containing 4 nM MCAK was added, resulting in the depolymerization of microtubules at 1.5 µm•min-1. Video playback is 100x real-time. (MOV 6016 kb)
Supplementary Video 2
This movie shows MCAK–GFP molecules (green) diffusing along microtubules (red) in 1 mM ATP. 0.5 nM MCAK-GFP was used. The TIRF images (FITC) were recorded in continuous mode at 100 ms per frame and overlaid onto one static epifluorescence image (TRITC) of the microtubules. Video playback is in real-time. (MOV 6039 kb)
Supplementary Video 3
This movie shows MCAK–GFP molecules (green) diffusing along microtubules (red) in 1 mM ADP. 0.5 nM MCAK-GFP was used. The TIRF images (FITC) were recorded in continuous mode at 100 ms per frame and overlaid onto one static epifluorescence image (TRITC) of the microtubules. Video playback is in real-time. (MOV 8912 kb)
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Helenius, J., Brouhard, G., Kalaidzidis, Y. et al. The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends. Nature 441, 115–119 (2006). https://doi.org/10.1038/nature04736
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DOI: https://doi.org/10.1038/nature04736
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