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