Using electrical and optical tweezers to facilitate studies of molecular motors

Abstract

Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells.

Fig. 1

A schematic depiction of a dielectrophoretically positioned and tightened actin filament suspended across a trench between two gold electrodes. A myosin-coated bead is being positioned near the filament with optical tweezers.

Fig. 2

(a) A series of bright-field optical micrographs of a myosin V-coated bead traveling along a tightly suspended actin filament. The illuminating condenser is stopped down from its maximum to increase contrast. The viewing objective is located below the sample. The time interval between frames is 850 ms and the exposure time is 80 ms. For clarity, dotted lines are overlaid on the actin filament. (b) Images of a stationary bead on the microscope slide surface moved in 150 nm increments along the optical axis, z. Images 1 and 8 are, respectively, closer to and farther from the imaging objective.

Fig. 3

(a) and (b) Smoothed surface plots of images similar to those in Fig. 2. (c) and (d) Fits of eqn (1) to images (a) and (b), respectively. (e) and (f) Average radial line profiles from the center of the images shown in (a) and (b), respectively (symbols), and the corresponding radial slices of the axisymmetric fits shown in (c) and (d), respectively (solid lines).

Fig. 4

(a) The displacement in the transverse direction, x (solid line) and the displacement in the optical axis, z (dashed line) of a myosin-V-coated bead, vs. the position along the filament, y. The z-position lags the x-position by roughly 90°, as expected for a left-handed helical path. (b) The left-handed, helical motion of the same myosin-V-coated bead as it traveled along an actin filament.