Investigating actin behavior using this protocol is significant because it allows the user to probe motor protein ensemble dynamics using the precision of optical trapping. The main advantage of this technique is the ability to measure myosin mechanics within actin structural hierarchy, as opposed to the traditional single-motor single-filament orientation used in many optical trapping experiments. Another advantage of this protocol is its modular nature, which allows the user to customize the assay to the hierarchical cytoskeleton system of their choosing.
To begin, apply two pieces of double-sided sticky tape to the middle of a microscope slide three to four millimeters apart from each other. Then tear or cut off the excess tape that hangs off the edge of the slide. Next, add the PLL-coated cover slip on top of the tape perpendicular to the long axis of the microscope slide to form a channel.
Using a small tube to compress the cover slip onto the tape and microscope, slide thoroughly until the tape is transparent, ensuring that there are no bubbles in the tape as this can cause leakage from the flow channel. Add 15 microliters of the diluted rhodamine actin to the PLL flow cell and wick the excess solution through the flow cell, but do not allow the flow channel to become dry. Then, incubate for 10 minutes in a humidity chamber.
Next, prepare a one milligram per milliliter casein solution in actin polymerization buffer and add 15 microliters of the solution to prevent non-specific binding of the subsequent components. Then, incubate for five minutes in a humidity chamber. Afterward, add the desired concentration of myosin to the prepared biotinylated actin and bead suspension and gently stir with a pipette tip.
Then, immediately add 15 microliters of the suspension along with the desired myosin concentration to the flow cell and incubate for 20 minutes. Next, seal the open ends of the flow cell with nail polish to prevent evaporation during imaging and optical trapping experiments. Once the system is ready, turn on the laser using the laser power button at the left bottom corner of the screen to 50 milliwatts and let it stabilize for 30 minutes.
Sequentially click on the illumination, camera, objective, and stage movement buttons within the software to bring up those windows for viewing and manipulation during the experiment. Turn the microscope illumination on by clicking on the On/Off button and setting it to maximum power by clicking and dragging the bar all the way to the right. Open the sample area and remove the sample holder from the microscope stage.
Then add the flow cell and secure it with the metal sample holders, ensuring that the cover slip is on the bottom. Afterward, add 30 microliters of RO water to the center of the bottom objective and reinsert the sample stage. Then raise the lower objective using the L2 on the remote controller until the bead of water touches the cover slip.
Next, lower the top objective until about half the distance to the flow cell is reached using the R2 on the remote controller. Then, add 170 microliters of RO water to the top of the flow cell directly under the top objective and lower the top objective until it breaks the surface tension of the water and forms a meniscus. Subsequently, move the microscope stage using the arrow pad on the remote controller until the edge of the tape adjacent to the flow channel is reached and then close the sample door.
Using the objective window in the screen, bring the edge of the tape and focus by bringing the bottom objective named Laser Objective up by clicking on the upper arrow and the top objective by clicking bottom arrow using the onscreen controls. Find a floating bead and trap it by clicking on the trap shutter button, which will open the shutter and allow the trapping laser to hit the sample. Then, click on the trap cursor on the screen and drag it to move the location of the trapping laser.
Once trapped, calibrate the bead by clicking on the calibration button. Then, click on Settings and type in the diameter of the bead and temperature of the stage found in the bottom left of the software window. Next, click on Trap 1, then on X Signal and perform the corner frequency fit by clicking on Run.
Then click and drag within the window to optimize the function fit. Afterward, click on Use it for sensitivity and stiffness values and then accept values. Following, close the window.
Find an actomyosin bundle by searching for beads bound to AFs on the surface of the cover slip and by looking for both fluorescent AFs co-localized. Turn on the white light source and use the appropriate filter cube to image each actin filament by turning the turret. Once verified, trap the bead attached to the top filament of the bundle by clicking on the trap shutter button.
Then, record the data by clicking on the oscilloscope button using the onscreen controls. To visualize measurements without recording the data, click on Start and then click on Autosave to save all the data. To record measurements, click on Start record and choose which data are to be visualized in real time by choosing from the dropdown menu X Signal or Y Signal.
The force trace of skeletal myosin motors generating force within the constructed in vitro actin structural hierarchy exhibit steady ramp in force until a plateau is reached over two to five minutes. However, it is also observed that some actomyosin bundles that do not generate any net force and the force traces appear as baseline noise or exhibit no substantial net increase in force over 90 seconds due to a low concentration of motor or an unfavorable parallel orientation of the filaments. This protocol development will pave the way toward building more complex in vitro cytoskeletal assays that will further help model large-scale cellular tasks, such as cell division and muscle contraction from the bottom up.
