Geometrical and temporal
factors determining kinesin-dependent
microtubule motility in vitro
Eberhard Unger1, Roland Stracke1, Jörg Burgold2, Hans-Joachim Schacht2, Konrad J. Böhm1
of Molecular Biotechnology, Jena
2Institute for Microsystem Technique, Mechatronics and Mechanics, Technical University Ilmenau, Germany
Eukaryotic cells synthesize specialized motorproteins
interacting with 25-nm thick microtubules and converting
chemical energy of nucleoside triphosphates into mechanical force,
required for different intracellular transport processes. A prominent motorprotein
is kinesin, which is able to mimic cellular motility
vitro driving taxol-stabilized microtubules across kinesin-coated surfaces.
This model system might provide a basis for future developments of nanoscaled
machineries. The present study describes geometrical and temporal
factors affecting kinesin-dependent microtubule motility.
First, the surface roughness tolerable for microtubule gliding was determined, using polished silicon wafers into which defined steps were etched. Kinesin-driven microtubule gliding across the wafer surface was visualized by video-enhanced DIC microscopy in reflected light mode. The maximum step height which microtubules were able to overcome upwards was found to be about 280 nm.
Second, microtubule gliding was investigated between two glass slides to determine the minimal distance, which still allows gliding. For this reason, microtubules were transferred to a kinesin-coated glass slide. To produce clefts of variable height, a slightly curved coverslip was approximated (convex side down) to the slide by means of a small vacuum chamber. An interferometric method was used to measure the distance between both glass surfaces: Around the attachment point of both glasses Newton fringes appear, which were visualized using reflected light mode. After defining the area of interest in relation to the interference fringes, microtubule gliding was observed by video-enhanced DIC or phase contrast microscopy. The actual distance between both glasses is given by the theory of the Newton fringes. The velocity of microtubule gliding was found to depend on the gap height between both glasses. Motility was observed down to a distance of about 100 nm. In a single case, a microtubule migrated into a gap of 30 nm, where its leading edge stuck and the other part showed a fishtailing-like movement.
Third, the maximal time was determined which the system was working under standard conditions. In a sealed chamber, microtubules moved over a period of 3 hours with nearly constant velocity. Basing on the standard velocity of 600-800 nm/s, a microtubule can theoretically migrate a distance of 6.5-8.6 mm within this time interval. To study whether the microtubules were attached to the surface all the time, the path of individual microtubules was followed by displacement of the microscope stage to keep them in the observation field. The microtubules observed migrated over distances up to about 1 mm without detaching from the surface. Thereafter, they were lost either by inaccurate stage displacement or by impaired imaging. As only linear tracks could be measured and the microtubules migrated along curved ones, the actual path length should be still greater.
Our results demonstrate that kinesin-driven microtubules, which can be regarded as biomolecular nanomotoric units, are able to work continuously up to several hours on uneven surfaces with height differences up to 280 nm and in narrow chambers down to about 100 nm height. Further studies are necessary concerning the unipolar alignment of the microtubules and stabilization of the components involved in force generation.