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Catalysis of the microtubule on-rate is the major parameter regulating the depolymerase activity of MCAK

Abstract

The kinesin-13, MCAK, is a critical regulator of microtubule dynamics in eukaryotic cells. We have functionally dissected the structural features responsible for MCAK's potent microtubule depolymerization activity. MCAK's positively charged neck enhances its delivery to microtubule ends not by tethering the molecule to microtubules during diffusion, as commonly thought, but by catalyzing the association of MCAK to microtubules. On the other hand, this same positively charged neck slightly diminishes MCAK's ability to remove tubulin subunits once at the microtubule end. Conversely, dimerization reduces MCAK delivery but improves MCAK's ability to remove tubulin subunits. The reported kinetics for these events predicts a nonspecific binding mechanism that may represent a paradigm for the diffusive interaction of many microtubule-binding proteins.

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Figure 1: MCAK mutants have shifted microtubule depolymerization dose-response curves.
Figure 2: Diffusive behavior of single MCAK molecules and mutants.
Figure 3: A positive correlation between kON and kOFF is consistent with an energy-landscape model for diffusive binding.

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Acknowledgements

The authors acknowledge support from the National Institutes of Health (GM069429) and from the National Science Foundation (IGERT traineeship to J.C.).

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Authors

Contributions

J.R.C. built the TIRF microscope, developed the coverslip coating and assay conditions, purified some of the protein samples, wrote the analysis software, performed all of the experiments and wrote the manuscript. M.W. engineered the DNA constructs and purified and calibrated some of the protein samples. C.L.A. assisted with the TIRF configuration. L.W. oversaw the entire project, suggested experiments and assisted with data interpretation and also with the writing of the manuscript.

Corresponding author

Correspondence to Linda Wordeman.

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Supplementary Table 1 and Supplementary Figures 1 and 2 (PDF 283 kb)

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Cooper, J., Wagenbach, M., Asbury, C. et al. Catalysis of the microtubule on-rate is the major parameter regulating the depolymerase activity of MCAK. Nat Struct Mol Biol 17, 77–82 (2010). https://doi.org/10.1038/nsmb.1728

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