Abstract
Kinesin and cytoplasmic dynein are microtubule-based, force-generating ATPases thought to be involved in the bidirectional transport of vesicular organelles along microtubules. In vitro, kinesin and dynein promote movement of 0.1 - 0.2μm plastic beads toward the plus and minus ends, respectively, of microtubules. In these systems, the proteins are thought to remain fixed, by nonspecific absorption, to the bead surface. The motion of the bead on the microtubule is detected by video enhanced, DIC microscopy, and reflects the movement of the motor protein along the microtubule.To quantitate bead motion, a method was devised for tracking the position of kinesin or dynein coated beads moving along microtubules. The method involves 1) cross-correlation of a template bead image (the kernal) with each of the frames in a video sequence, followed by 2) calculation of the centroid of the cross-correlation function for each of the frames in the sequence. The centroid provides a measure of bead position. Application of this method to the measurement of the relative position of two stationary beads adsorbed nonspecifically to the glass surface indicates that the standard deviation of the measured horizontal and vertical coordinates can be less than 0.5 nm. More recently, we have determined that centroid calculation of the unprocessed bead image (i.e. without cross-correlation) can provide a measure of bead position with standard deviations in the horizontal and vertical directions of less than 2 nm. The latter method is faster, but more sensitive to the contrast and magnification of the video image.