The distance that kinesin-1 holds its cargo from the microtubule surface measured by fluorescence interference contrast microscopy.
Kinesin-1 is a motor protein that carries cellular cargo such as membrane-bounded organelles along microtubules (MTs). The homodimeric motor molecule contains two N-terminal motor domains (the motor "heads"), a long coiled-coil domain (the "rod" or "stalk"), and two small globular "tail" domains. Much has been learned about how kinesin's heads step along a MT and how the tail is involved in cargo binding and autoinhibition. However, little is known about the role of the rod. Here, we investigate the extension of the rod during active transport by measuring the height at which MTs glide over a kinesin-coated surface in the presence of ATP. To perform height measurements with nanometer precision, we used fluorescence interference contrast microscopy, which is based on the self-interference of fluorescent light from objects near a reflecting surface. Using an in situ calibrating method, we determined that kinesin-1 molecules elevate gliding MTs 17 +/- 2 nm (mean +/- SEM) above the surface. When varying the composition of the surrounding nucleotides or removing the negatively charged -COOH termini of the MTs by subtilisin digestion, we found no significant changes in the measured distance. Even though this distance is significantly shorter than the contour length of the motor molecule ( approximately 60 nm), it may be sufficient to prevent proteins bound to the MTs or prevent the organelles from interfering with transport.
Kerssemakers
Jacob
J
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Howard
Jonathon
J
Hess
Henry
H
Diez
Stefan
S
D000818:Animals; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference; D008870:Microtubules / Q000378:metabolism*; D013379:Substrate Specificity
D029721:Drosophila Proteins; C515209:Khc protein, Drosophila; D016547:Kinesins