Abstract
Determination of the number of active motors pulling a single MT or bead during motility assays has proven difficult. Traditional protein concentration assays, such as Bradford, cannot distinguish between active and inactive motors. We attach a superparamagnetic bead to the (+) end of a microtubule. When placed in a magnet with uniform magnetic field gradient, the bead pulls on the MT with a controllable 0-10 pN force. If the force is perpendicular to the gliding direction of the MT, a short section of the MT “pops off” the surface every 2 to 5 s, as shown in the diagram. This detachment is characterized by rapid motion of the superparamagnetic bead in the direction of higher magnetic field gradient followed by normal microtubule gliding velocity when the MT is pulled taut. The length of the short section between “popoffs” is the distance between active kinesins along a microtubule.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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