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, enabl