Reconstructing the Motility Driven by Membrane-Bound Myosin on the Inner Surface of Cell-Sized Droplets

Molecular motors and the cytoskeleton perform essential cellular functions by interacting with the cell membrane. Reconstituting the behavior of motor proteins interacting with biological membranes and cytoskeletal filaments within a cell-sized space provides insights into their intracellular functions. A water-in-oil (W/O) emulsion droplet represents an invaluable experimental system because it offers an encapsulated space that mimics the intracellular environment. In this study, we aimed to reconstitute the actomyosin motility on the flat membrane surface of cell-sized W/O droplets using membrane-bound myosin I that anchors to and exerts force on both the cell membrane and actin cytoskeleton. Myosin IC or myosin ID, which binds specifically to the cell membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] together with the actin cytoskeleton, was encapsulated within W/O droplets. Myosin IC and myosin ID caused gliding of actin filaments on the inner bottom membrane of the hemispherical droplet containing PI(4,5)P₂, and myosin-driven actin filament movement on the flat membrane surface was quantified. Fast motor myosin ID was more sensitive than slow motor myosin IC to the geometrical conditions and binding manner to the membrane. Therefore, the <i>in-droplet</i> actin filament gliding assay is a useful tool for elucidating the molecular mechanisms underlying cellular events occurring at the cell membrane, which are achieved through the concerted action of myosin and the actin cytoskeleton.