Study cytoskeletal motors in real-time at the nanoscale

In this assay, we investigated the displacement of actin with respect to wild-type myosin-VI by measuring the correlated displacements of the two beads trapped in the dual optical trap. Figure 1 shows that myosin pulls the actin filament in a unidirectional manner with a measurable force.

The exact kinetics of the motor can be observed by measuring the relative translocation of the filament with respect to the motor and the related forces. From the resulting force–time traces, we can derive the speed and processivity of the motor.

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Sample courtesy of Prof. Dr. Margaret Titus at the University of Minnesota

We can follow the motor’s activity in all 3 dimensions as it moves over the filament and measure the distance, velocity, and (stall-) force of a motor attached to a bead, also under the influence of other molecules and loads.

Figure 2 shows a single kinesin motor stepping along a microtubule. Steps, 8 nm in size, were collected with 2D feedback along the microtubule direction at stall force of 1 pN.

In Figure 3 we can observe 2 events during which the kinesin motor detaches from the microtubule at a force of approximately 2 pN.

Data courtesy of Dr. Paul Ruijgrok and Dr. Zev Bryant at Stanford University.

The measured data in Figures 1 and 2 show the extension of a vimentin intermediate filament. The force-distance curve is measured while stretching and relaxing the intermediate filament at a slow speed, allowing for structural equilibrium. The retraction curve shows clear hysteresis due to the remodeling of the vimentin filament under high tension. Simultaneous confocal fluorescence imaging of the vimentin filament is used to resolve this intra-molecular remodeling.

Combining optical tweezers with simultaneous fluorescence measurements allows correlating the mechanical properties of the microtubule with local information.

Figure 1 shows the interaction between two fluorescently labeled microtubules, as one microtubule is dragged across the other with a known force. In the middle and left snapshot, we can observe that the friction force between the filaments causes one microtubule to adopt a curved conformational structure. When we stop the movement of the microspheres the microtubule slowly relaxes (right).

The surface properties of these filaments, the electrostatic and ionic forces and specific interactions, can generate friction when two filaments interact. This friction can be measured using a device such as the C-Trap^® configured for quadruple optical trapping.