A method for reconstituting the motility of membrane-bound myosin on the surface of the cell-sized W/O droplet.

Membrane-bound myosin generates force through interactions with the cytoskeletal actin filament beneath the cell membrane and constitutes the mechanical basis for living cells. Myosin ID, a membrane-bound myosin, drives the gliding motion of actin filaments and binds to phospholipids in the lipid membrane of a living cell. Here, we describe the in-droplet actin filament gliding assay, a method that reconstitutes the motility of actomyosin that dynamically interacts with lipid membranes within water-in-oil (W/O) droplets, which mimic the confined geometry of the intracellular environment. Our method enables quantification of the gliding velocity of actin filaments driven by myosin ID on the inner surface of W/O droplets surrounded by a phospholipid membrane. The in-droplet actin filament gliding assay provides a valuable platform for reconstituting the motile properties of other membrane-bound myosins on membrane surfaces in confined spaces and for analyzing the dynamics of actomyosin networks. The main features and applications of this method are as follows:•Reconstitution of actin filament gliding driven by membrane-bound myosin ID within confined water-in-oil droplets.•Quantitative evaluation of actomyosin dynamics on the inner surface of the lipid membrane within water-in-oil-droplets.•Broadly applicable assay platform for studying the motile properties of membrane-associated myosin families.