Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy is a single molecule technique based on laser tweezers that makes it possible to investigate the chemomechanics of myosins motors under load with the highest time resolution. This technique allows probing very rapid events in force production by maintaining the force constant during every phase of the motor interaction with full control on force directionality. By making appropriate adjustments, this protocol could be easily applied to other kinds of processive motors, including kinesins and dyneins, to uncover the regulation by force.

After mounting one set of acousto-optic deflectors onto a linear translator, take an iris and adjust its aperture to fit the objective back aperture size. Replace the objective with the iris centered on the threaded objective housing. Move the translator toward the laser beam until the portion of the beam blocked by the piezo crystal is visible after the iris, and turn the translator slightly backward until the beam completely fills the iris aperture.

To prepare silica beads in pentyl acetate, first dilute 20 microliters of 1.2 micron 10%solid silica beads in 1-1.5 milliliters of acetone with vortexing and 30 seconds of sonication. After sonicating, collect the beads by centrifugation and resuspend the bead pellet in 1 milliliter of fresh acetone for a second wash. After the second acetone wash, wash the beads three times in 1 milliliter of fresh pentyl acetate per wash under the same centrifugation conditions, and resuspend the pellet in 100 microliters of 1%of nitrocellulose and 900 microliters pentyl acetate.

Next, use a pure ethanol-soaked piece of lab tissue to carefully wipe a 24 x 24 millimeter glass coverslip before using clean tweezers to rinse the glass directly in the ethanol and allowing the coverslip to dry under gentle nitrogen flow. Vortex and sonicate the prepared silica bead stock for 30 seconds and use an ethanol-cleaned 24 x 60 millimeter coverslip to smear 2 microliters of silica bead solution onto the surface of the smaller coverslip. While the beads are drying, clean a 26 x 76 millimeter slide as demonstrated, and place two 60-100 micron-thick lines of 3 millimeter double-sided taped onto the long edges of the slide.

Using clean tweezers, place the bead-coated coverslip onto the tape with the beads facing the inside of the slide chamber, and fill the chamber with 15 millimolar phosphate buffer and floating polystyrene bead solution. Then, seal the chamber with silicon grease. To calibrate the pixel-to-nanometer ratio of the brightfield camera, acquire a first image of a silica bead on the coverslip surface approximately 5 microns from the center of the field of view.

Move the stage to displace the bead 10 microns toward the center of the field of view and take a second image. Calculate the camera calibration constant by measuring the pixel distance between two positions of the bead. To calibrate the trap position, trap a single floating particle in one trap and move the acousto-optic deflectors in 0.2 megahertz steps, acquiring an image of the particle and the corresponding frequency of the deflectors for each step.

Then, repeat the calibration for the second trap in the same manner. To calibrate the trap power and stiffness, trap a single particle in each trap and displace both traps using the acousto-optic deflectors in 0.2 megahertz steps, recording a Brownian motion of the particle in both traps with Quadrant Photodiode Detectors and the corresponding frequency of the acousto-optic deflectors. Obtain calibration constants by fitting a Lorentzian function to a power spectrum of the recorded Brownian motion.

To assemble a sample for measurement, add 1 milligram per milliliter of biotinylated BSA to a new chamber with silica beads on the coverslip surface. After 5 minutes, wash the chamber with AB buffer and add 1 milligram per milliliter of streptavidin to the chamber. After 5 minutes, wash the chamber as demonstrated, and add 3 nanomolar biotinylated Myosin-5B heavy meromyosin in M5B buffer supplemented with 2 micromolar Calmodulin.

After 5 minutes, wash the chamber three times with 1 milligram per milliliter of biotinylated BSA supplemented with 2 micromolar Calmodulin in AB buffer and wait 3 minutes after the final wash. Flow the reaction mixture and seal the chamber with silicon grease. To measure the sample, place the chamber onto the microscope stage and move the objective until the floating alpha-actinin beads are in focus.

Switch on one trap and trap one bead. Once the first trap is occupied, move the translator to position the trapped bead close to the coverslip surface to avoid trapping multiple beads, and trap a bead in the second trap. Displace the sample through the long-range translators to locate actin filaments of at least 5 microns in length within the solution.

When a filament has been spotted, move the sample to let a trapped bead approach one end of the filament. Once the filament has been attached, adjust the bead's distance to the approximate filament length and move the stage to create a flow in the unbound bead direction. The filament will become stretched by the flow and eventually bind to the second bead.

The resulting complex is called a dumbbell"To establish actin-myosin contact, gently separate the two traps and set the filament to pretension levels up to about 3 piconewtons. To probe the rigidity of the dumbbell, make one trap oscillate in a triangular wave by changing the frequency of one acousto-optic deflector and verifying the consequent transmission of the motion to the trailing bead through its position signal. Move the stage to position the dumbbell in the proximity of a pedestal silica bead and adjust the height of the dumbbell so that the actin filament gets in contact with the silica bead surface.

As observed in this representative position record, when the myosin motor is not interacting with the actin filament, the trapped beads move at a constant velocity against the viscous drag force of the solution. Once the myosin motor begins interacting with the filament, the force carried by the moving filament is very rapidly transferred to the protein, and the system velocity drops to zero, with the stepping events occurring under constant force till the end of the run. The force is switched from the positive to the negative direction by the feedback system, which switches the force direction when the bead reaches the edge of the oscillation range set by the user.

When the myosin binds and displaces the filament toward the positive direction, it pushes the bead toward the upper edge of the oscillation range. If this happens under assistive force, the run of the myosin will be interrupted by the force direction inversion at the oscillation edge, limiting the length of the run to the amplitude of the dumbbell oscillation. Precise alignment of the optical system is necessary to achieve the optimal spatial and temporal resolution, while an accurate calibration is necessary to precisely determine the values of the applied forces.

This technique has been adapted to study the sliding and targeted search of transcription factors in DNA as well as dynamic interactions between mechanotransduction proteins inducting over microtubule filaments.