To begin, add one chamber volume of purified human myosin-7a protein in 500 millimolar sodium chloride motility buffer to the flow chamber, and incubate it for five minutes at room temperature. Wash the flow chamber with three chamber volumes of one milligram per milliliter BSA in 150 millimolar sodium chloride motility buffer. After that, draw the liquid by using a clean wipe at the outlet to absorb excess liquid.
Then, flow one chamber volume of 10 nanomolar rhodamine-phalloidin-labeled F-actin in 150 millimolar sodium chloride motility buffer through the flow chamber. Monitor the binding of actin filaments to the surface under a fluorescent microscope with a 100x objective. Ensure optimal actin filament density for tracking, with enough filaments in the field of view but sparse enough to prevent overlapping.
Next, wash the flow chamber with three chamber volumes of 150 millimolar sodium chloride motility buffer to remove unbound actin filaments and excess rhodamine-phalloidin. To initiate motility, add one chamber volume of the final buffer to the flow chamber. Then, record images on a fluorescence microscope using 561-nanometer excitation to visualize rhodamine-phalloidin-labeled actin.
Find an area on the slide with the desired actin density and capture images every 30 seconds for 30 minutes. Next, download and install the Fast program from the GitHub repository. Ensure that it is the Python 3-compatible version with an additional module for ND2 file conversion.
Run the Fast program using a tolerance value of 33 to retain only smoothly moving filaments. Afterward, use the Fast program to analyze the newly created TIFF images. Set the x maximum value to 20, 000 nanometers and the y maximum value to 20 nanometers per second to represent the longest filament length and maximum filament velocity.
Then, use px to set the pixel size of the acquired image to 65 nanometers. Set minV to 0.1 nanometers per second as the minimum velocity required for filament inclusion in the analysis. To set the maximum distance allowed between frames for filaments to be linked, use maxD, avoiding the linking of separate filaments between frames.
Use pt to set the tolerance for fluctuations in filament velocities, including only those filaments with smooth movements. Lastly, use d to indicate the folder containing the TIFF stacks for analysis. The actin filament gliding assay demonstrated myosin-7a's slow motility, with the video playback sped up 500 times to visualize the movement.
The velocity distribution of myosin-7a showed a peak below five nanometers per second, confirming its slow motility.
