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Our current interest is to understand how the microtubule motor protein yeast cytoplasmic dynein produces force, to dissect its force-producing mechanical elements and to analyze its stepping behavior as a function of opposing as well as assisting loads. Measurements are performed with a custom-built optical trapping microscope. Our setup is primarily based on the optical tweezers setup described in detail by Mehta et al. (Methods Enzymol. 1998, 298:436-459) and Rice et al. (Methods Enzymol. 2003, 361:112-133) and further enhanced by a force-feedback mode, which allows accurate step size measurements under constant average loads.
A schematic representation of the optical trapping assay is shown in Figure 1. Here, the dynein motor is coupled to an anti-GFP antibody-coated 1 um latex bead through an N-terminal GFP-tag on the motor tail. The dynein-coated bead is than captured by the optical trap and positioned above an axoneme (bundles of microtubules from sea urchin flagella) or microtubule attached to the glass surface. Upon microtubule binding, cytoplasmic dynein moves processively (ability to take multiple steps without dissociation) towards the microtubule-minus-end (Figures 2 and 3A).
Fig. 2. Example optical trapping record of a single full-length yeast cytoplasmic dynein molecule at 1 mM ATP showing motor stalling (trap stiffness: k = 0.049pN/nm). The axoneme and trap remained fixed during the measurement (non-feedback mode) (note that the record appears highly compressed due to the slow dynein movement (<50 nm/s) and the 2 kHz sample rate).
Fig. 3. Optical trapping measurement of cytoplasmic dynein stepping under a constant rearward load of 3 pN (force-feedback mode). (A) Bead position (black trace) and trap position (green trace) as a function of time (trap stiffness: k = 0.049pN/nm). (B) Average bead-trap separation (upper inset) and corresponding average force load (F = -kx) (bottom inset). (C) Example trace segments showing motor steps. The raw data are shown in black and the steps detected by a step finding program (Kerssemakers et al., 2006, Nature 442, 709-712) in red (advancing mode) and blue (non-advancing mode).
The dynein-coated beads move away from the center of the optical trap (non-feedback mode) and than eventually stall at an average rearward load of ~7 pN (Figure 2). The force load that the motor experiences increases with every step that the motor advances due to the increasing bead-trap separation (the load is proportional to the distance between the trap and bead center).
mode of the optical trap is used to measure the stepping behavior of motor proteins
as a function of a constant force load. Here, the optical trap maintains a constant
average bead-trap separation (Figure 3B, upper inset) to generate a constant
average force load (Figure 3B, bottom inset). Under these conditions, discrete
steps of cytoplasmic dynein can be measured (Figure 3C, 3 pN rearward load).
Unlike kinesin, which shows a regular, unidirectional stepping behavior (Figure
4), dynein displacement records display a mixture of forward as well as backward
steps (Figure 3C).
|Fig. 4. Example optical trapping record of processive movement of a single K560-GFP molecule (Tomishige et al., 2002, Science 297:2263-2267) at 1 mM ATP and 6 pN rearward load (trap stiffness: k = 0.068pN/nm). The area in which the motor experiences a constant average load from the feedback-controlled optical trap is indicated by the grey shaded background (±200 nm).|
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