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Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain

Abstract

Conventional kinesin transports membranes along microtubules in vivo, but the majority of cellular kinesin is unattached to cargo. The motility of non-cargo-bound, soluble kinesin may be repressed by an interaction between the amino-terminal motor and carboxy-terminal cargo-binding tail domains, but neither bead nor microtubule-gliding assays have shown such inhibition. Here we use a single-molecule assay that measures the motility of kinesin unattached to a surface. We show that full-length kinesin binds microtubules and moves about ten times less frequently and exhibits discontinuous motion compared with a truncated kinesin lacking a tail. Mutation of either the stalk hinge or neck coiled-coil domain activates motility of full-length kinesin, indicating that these regions are important for tail-mediated repression. Our results suggest that the motility of soluble kinesin in the cell is inhibited and that the motor becomes activated by cargo binding.

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Figure 1: Domain organization of wild-type and mutant kinesin constructs.
Figure 2: Tracking the movement of single kinesin molecules.
Figure 3: Histograms of velocities for single fluorescent kinesin molecules.

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Acknowledgements

We acknowlege the members of the Vale laboratory for their help in many aspects of this work. We thank J. Hartman, S. Hopkins, D. Pierce, A. Rudner and K. Thorn for insightful discussion, experimental help, and assistance with the manuscript; C. Hart and J. Ubersax for preparing the K560 (neck mut.) clone; L. Lachman and Y. Cabeza-Alvelaiz for providing the kinesin light chain clone and technical advice. D.S.F. is supported by UCSF Cell Biology Training Grant number T32 GM08120.

Correspondence and requests for materials should be addressed to R.D.V. The protein sequences for human ubiquitous kinesin heavy chain and light chain have been deposited at the Protein DataBank under accession numbers U06698 and L04733, respectively.

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Friedman, D., Vale, R. Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain. Nat Cell Biol 1, 293–297 (1999). https://doi.org/10.1038/13008

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