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

Figures

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  Fig. 1. Silane coating procedure for glass coverslips. (a) Glass coverslips and glass rack. (b) Glass rack in 300 mL beaker. (c) Glass coverslips within silanization solution I in a petri dish.

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  Fig. 2. Silane-coated glass flow chamber. To construct the chamber, strips of double-sided tape were placed on a silane-coated glass coverslip at 1-mm intervals to serve as spacers. A non-treated glass coverslip was then placed on top, and the areas in contact with the tape were lightly pressed to form the flow chamber. The resulting flow chamber had a volume of approximately 3 μL.

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  Fig. 3. Construction of emulsion containing W/O droplet. (a) To construct W/O droplets, the 600 µL tube containing the aqueous phase and lipid-in-oil mixture was vigorously rubbed against a metal tube rack in the direction indicated by the white arrow. (b) Tube containing the aqueous phase and lipid-in-oil mixture before rubbing (left) and tube containing the emulsion after rubbing (right). Within the tube, the aqueous phase and lipid-in-oil mixture were mixed, resulting in the formation of W/O droplets.

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  Fig. 4. Image of in-droplet actin filament gliding assay. (a) A field of view of the in-droplet actin filament gliding assay. The aqueous phase of W/O droplets contained numerous actin filaments formed by the polymerization of G-actin monomers. Actin filaments were located at the inner surface of the W/O droplet via membrane-bound myosin ID. Most actin filaments successfully exhibited gliding motion induced by myosin ID on the inner surface of the W/O droplets. Scale bar, 20 µm. (b) Schematic of the in-droplet actin filament gliding assay.

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  Fig. 5. Quantification of gliding actin filament velocity. (a) Box plot showing the gliding velocity of actin filaments driven by myosin ID. The box represents the 25th–75th percentiles, and the mean value is indicated by a black line. The mean gliding velocity was 178 ± 26 nm·s−1 (mean ± standard deviation; n = 32 actin filaments). (b) Bar graph showing the fraction of gliding (blue) and stalled (grey) actin filaments on the inner surface of W/O droplets. The mean fraction of gliding actin filaments was 0.9 ± 0.1 (mean ± standard deviation; n = 6 droplets).