The Gliding Actin Filament Assay: An In Vitro Motility Assay to Study Translocation of Actin Filaments on Immobilized Myosins
Transcript
Myosin – a motor protein – interacts with another filamentous protein, actin, causing its translocation.
To analyze the gliding movement of actin, assemble a glass slide-based flow chamber such that the inner portion of the chamber-top is nitrocellulose-coated. Flow in the myosin proteins through the chamber, which immobilize over the nitrocellulose coating.
Supplement the chamber with unlabeled actin filaments – black actins – and add a motility buffer containing adenosine triphosphate or ATP molecules.
The actin filaments bind to immobilized active myosins along with inactive or dead myosins. ATP molecules interact with the active myosins, leading to their dissociation from black actins while the inactive myosins remain actin-bound.
Centrifuge to pelletize inactive myosins attached to black actins. Rinse with a buffer thoroughly to remove the inactive myosin pellet, free actin filaments, and unused ATPs. Flow in fluorescently-labeled actins that bind immobilized, active myosins.
Supplement the chamber with a final buffer containing a crowding agent – methylcellulose, and ATP. The crowding agent forces actin filaments to remain closer to the chamber surface.
Each active myosin hydrolyzes ATP leading to conformational change and binding to a new position along the filament, generating a power stroke that glides the actin over myosin.
Observe the chamber under a fluorescence microscope to record the motion of labeled actin filaments gliding smoothly over the immobilized myosin heads, confirming successful translocation.
