Direct observation of single kinesin molecules moving along microtubules.
Kinesin is a two-headed motor protein that powers organelle transport along microtubules. Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule. Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead). The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a approximately 1% probability of detachment per mechanical cycle. Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl. Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism.
Vale
R D
RD
Yanagida BioMotron Project, ERATO, JRDC, Senba-Higashi, Osaka, Japan.
Funatsu
T
T
Pierce
D W
DW
Romberg
L
L
Harada
Y
Y
Yanagida
T
T
D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila; D004926:Escherichia coli; D006801:Humans; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016547:Kinesins