Figure 1.

Theoretical predictions for the TCM assay. (a) A schematic of the in vitro motility assay is shown, which uses 0.5 μm beads conjugated to the MT-dependent motor molecule via its natural tail extension or an artificial elongated link. The MT is immobilized on the coverslip; the red arrow indicates the direction of motor walking in the stationary coordinate system XYZ. (b) Predicted XY coordinates are shown for the bead moving on top of the MT at 20 μm min⁻¹ in the stationary coordinate system (500 coordinates for each tether with PL = 46 nm). Lines representing MTs are shown in dark red. (c) A schematic of motor-MT attachment in the moving system of coordinates is shown. Axis X’ points along the MT in the direction of motor walking (black arrow), Y’ and Z’ are perpendicular to the MT, and the origin is located on the surface of the MT at the attachment site for motor domains. Two example attachments to different protofilament tracks are shown. Angle α measures the position of the walking motor relative to the protofilament on top of the MT, for which α = 0°. (d) Two-dimensional “cloud” plots show the predicted bead excursions for 30 nm (blue) and 200 nm (red) tethers in the moving coordinate system. Data are for α = 0°; there is a total of 2000 points for each tether with PL = 46 nm. (e) The predicted relationship between the SD of MT-perpendicular bead excursions along the Y’-axis (Y’-excursion) and contour lengths for different tethers are shown. The lines are empirical fits with the following functions: a + bx + cx^0.5. Data for each tether is based on n = 9000 coordinates. Error bars (SD) were generated by bootstrap analysis; see Materials and Methods. To see this figure in color, go online.