Visualisation of a kinesin-13 motor on microtubule end mimics.
An expanding collection of proteins localises to microtubule ends to regulate cytoskeletal dynamics and architecture by unknown molecular mechanisms. Electron microscopy is invaluable for studying microtubule structure, but because microtubule ends are heterogeneous, their structures are difficult to determine. We therefore investigated whether tubulin oligomers induced by the drug dolastatin could mimic microtubule ends. The microtubule end-dependent ATPase of kinesin-13 motors is coupled to microtubule depolymerisation. Significantly, kinesin-13 motor ATPase activity is stimulated by dolastatin-tubulin oligomers, suggesting, first, that these oligomers share properties with microtubule ends and, second, that the physical presence of an end is less important than terminal tubulin flexibility for microtubule end recognition by the kinesin-13 motor. Using electron microscopy, we visualised the kinesin-13 motor-dolastatin-tubulin oligomer interaction in nucleotide states mimicking steps in the ATPase cycle. This enabled us to detect conformational changes that the motor undergoes during depolymerisation. Our data suggest that such tubulin oligomers can be used to examine other microtubule end-binding proteins.
Moores
Carolyn A
CA
School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK. c.moores@mail.cryst.bbk.ac.uk
Milligan
Ronald A
RA
D000227:Adenine Nucleotides / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D047630:Depsipeptides / Q000737:chemistry; D016547:Kinesins / Q000737:chemistry*; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry*; D008958:Models, Molecular; D010961:Plasmodium / Q000378:metabolism*; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry*; D014404:Tubulin / Q000737:chemistry
D000227:Adenine Nucleotides; D047630:Depsipeptides; D008869:Microtubule-Associated Proteins; D015800:Protozoan Proteins; D014404:Tubulin; D000251:Adenosine Triphosphatases; D016547:Kinesins; C064570:dolastatin 10