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

Area of Science:

• Cell Biology
• Biophysics
• Biochemistry

Background:

• Molecular motors and the cytoskeleton are crucial for cellular functions, interacting with the cell membrane.
• Reconstituting these interactions in a cell-sized environment aids understanding of intracellular processes.
• Water-in-oil (W/O) emulsion droplets provide a cell-mimicking experimental system.

Purpose of the Study:

• To reconstitute actomyosin motility on the flat membrane surface of cell-sized W/O droplets.
• To investigate the behavior of membrane-bound myosin I interacting with the cell membrane and actin cytoskeleton.
• To analyze the force-exerting mechanisms of myosin motors on cytoskeletal filaments.

Main Methods:

• Encapsulation of myosin IC or myosin ID within W/O droplets containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2].
• Utilizing the flat membrane surface of hemispherical droplets for observing actin filament gliding.
• Quantifying myosin-driven actin filament movement on the membrane surface.

Main Results:

• Myosin IC and myosin ID induced gliding of actin filaments on the PI(4,5)P2-containing droplet membrane.
• Myosin-driven actin filament movement was successfully quantified on the flat membrane surface.
• Fast motor myosin ID exhibited greater sensitivity to geometrical conditions and membrane binding than myosin IC.

Conclusions:

• The in-droplet actin filament gliding assay is effective for studying cell membrane-associated molecular mechanisms.
• This system elucidates the concerted actions of myosin and the actin cytoskeleton in cellular events.
• Understanding these motor protein dynamics provides insights into intracellular functions at the cell membrane.