Inverted Motility Assay: An In Vitro Technique to Visualize Myosin Movement on Immobilized Actin Filaments

Myosin - a motor protein - has a globular head domain that interacts with actin - a filamentous protein. This interaction is essential for the translocation of myosin along the actin filaments. To study this binding in vitro, perform an inverted motility assay of myosin over surface-tethered actin.

To begin, assemble an assay chamber by placing a biotinylated polyethylene glycol - PEG-coated coverslip over a microscope slide such that the coated surface faces the inside, creating the top of the chamber.

Pass a solution containing avidin - a glycoprotein - through the chamber. Avidin binds to biotin with high affinity, which enhances the actin adhesion. Next, flow in biotin-tagged actin filaments labeled with a red fluorophore. The biotin ligand binds to avidin, immobilizing the filaments on the coverslip surface.

Finally, add green fluorescent protein-labeled myosin 5A proteins in a buffer containing adenosine triphosphate, or ATP, molecules. Dual-headed myosin 5A, containing a trailing and a leading head, interacts with the immobilized actin filaments. The trailing head of the myosin binds to an ATP and detaches from actin.

The hydrolysis of the ATP generates a power stroke, enabling the trailing head to bind to a position ahead of the leading head along the filament. Through successive power strokes, the myosin moves along the immobilized actin filament without detaching.

Using fluorescence microscopy, record the movement of the green myosin molecules on the red actin filaments.

Prepare the solutions for myosin 5a inverted motility assay, and keep them on ice.

Wash the chamber with 10 microliters of motility buffer with DTT. Flow in 10 microliters of 1 milligram per milliliter BSA in motility buffer with DTT. Repeat this twice, waiting a minute after the third wash. Use tissue or filter paper to wick the solution through the channel by gently placing the corner of the paper at the flow chamber exit.

Then, wash the chamber with 10 microliters of motility buffer with DTT three times. Flow in 10 microliters of the NeutrAvidin solution in motility buffer with DTT, and wait for one minute. Then, wash with 10 microliters of that solution three times.

Flow in 10 microliters of bRh-actin-containing motility buffer with DTT using a large-bored pipette tip, and wait for one minute. Then, wash with 10 microliters of motility buffer with DTT three times.

Flow in 30 microliters of final buffer with 10 nanomolar myosin 5a, and immediately load onto the total internal reflection fluorescence microscope. Begin recording once the optimum focus for TIRF imaging is found.