Bead movement by single kinesin molecules studied with optical tweezers.
Kinesin, a mechanoenzyme that couples ATP hydrolysis to movement along microtubules, is thought to power vesicle transport and other forms of microtubule-based motility. Here, microscopic silica beads were precoated with carrier protein, exposed to low concentrations of kinesin, and individually manipulated with a single-beam gradient-force optical particle trap ('optical tweezers') directly onto microtubules. Optical tweezers greatly improved the efficiency of the bead assay, particularly at the lowest kinesin concentrations (corresponding to approximately 1 molecule per bead). Beads incubated with excess kinesin moved smoothly along a microtubule for many micrometres, but beads carrying from 0.17-3 kinesin molecules per bead, moved, on average, only about 1.4 microns and then spontaneously released from the microtubule. Application of the optical trap directly behind such moving beads often pulled them off the microtubule and back into the centre of the trap. This did not occur when a bead was bound by an AMP.PNP-induced rigor linkage, or when beads were propelled by several kinesin molecules. Our results are consistent with a model in which kinesin detaches briefly from the microtubule during a part of each mechanochemical cycle, rather than a model in which kinesin remains bound at all times.
Block
S M
SM
Rowland Institute for Science, Cambridge, Massachusetts 02142.
Goldstein
L S
LS
Schnapp
B J
BJ
D000251:Adenosine Triphosphatases / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D002875:Chromosomes / Q000502:physiology; D016547:Kinesins; D007834:Lasers; D008722:Methods; D008870:Microtubules / Q000502:physiology; D008954:Models, Biological; D012822:Silicon Dioxide; D014404:Tubulin / Q000378:metabolism / Q000502:physiology; D014741:Video Recording
D014404:Tubulin; D012822:Silicon Dioxide; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins