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

Sep 29, 2026

Membrane-bound myosin generates force through interactions with actin filaments beneath the cell membrane and underlies cellular mechanics. This MethodsX article presents an in-droplet actin filament gliding assay in water-in-oil (W/O) droplets whose geometry mimics confined intracellular spaces. The assay reconstitutes actomyosin motion via myosin ID interacting dynamically with lipid membranes, enabling quantitative measurement of actin gliding velocity on the inner surface of phospholipid membranes.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages9

Reading Time23 min

AccessFree to download

Background

Myosin–actin motility and gliding assays

Geometry-mimicking platforms: supported lipid bilayers and W/O droplets

Method overview

In-droplet actin filament gliding assay concept

Quantitative readout of gliding velocity

Main features and applications

MethodsX 16 (2026) 103755 Contents lists available at ScienceDirect MethodsX journal [homepage:](homepage: www.elsevier.com/locate/methodsx)[ www.elsevier.com/locate/methodsx](homepage: www.elsevier.com/locate/methodsx) | A method for reconstituting the motility of membrane-bound myosin on the surface of the cell-sized W/O droplet | | | --- | --- | | Yusei Satoa,b,*, Rieko Sumiyoshi a, Masahito Hayashi c, Masahiko Yamagishi a,d, Junichiro Yajima a,d,e,*
a Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan b Institute for Extra-cutting-edge Science and Technology Avant-garde Research (X-star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan
c Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan
d Komaba Institute for Science, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan
e Research Center for Complex Systems Biology, Universal Biology Institute, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan | | | G R A P H I C A L A B S T R A C T | | | | | A R T I C L E I N F O Keywords: Myosin A B S T R A C T 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, For a published article:Yusei Sato, Rieko Sumiyoshi, Masahiko Yamagishi, Takeshi Haraguchi, Kyohei Matsuda, Suguru Sato, Kohji Ito, Junichiro YajimaReconstructing the Motility Driven by Membrane-Bound Myosin on the Inner Surface of Cell-Sized Droplets Langmuir, 41 (2025), pp. 10,077–10,084 [https://doi.org/10.1021/acs.langmuir.4c04123](https://doi.org/10.1021/acs.langmuir.4c04123) * Corresponding authors. E-mail addresses: [yuseisato@jamstec.go.jp](yuseisato@jamstec.go.jp) (Y. Sato), [yajima@bio.c.u-tokyo.ac.jp](yajima@bio.c.u-tokyo.ac.jp) (J. Yajima). [https://doi.org/10.1016/j.mex.2025.103755](https://doi.org/10.1016/j.mex.2025.103755) Received 15 October 2025; Accepted 9 December 2025 Available online 9 December 2025 2215-0161/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ([http://creativecommons.org/licenses/by/4.0/](http://creativecommons.org/licenses/by/4.0/)). Y. Sato et al. MethodsX 16 (2026) 103755 Actin Artificial cell Motility Cell membrane Phospholipid 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 ofW/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. | Specifications table | | | --- | --- | | Subject area | Biochemistry, Genetics and Molecular Biology | | More specific subject area | Motility assay for motor prote

FAQs

What method does the article introduce for studying membrane-bound myosin motility?

It describes an in-droplet actin filament gliding assay performed inside water-in-oil (W/O) droplets with a surrounding phospholipid membrane.

How does the assay quantify actomyosin behavior?

It enables quantitative evaluation of actin filament gliding velocity driven by myosin ID on the inner surface of the lipid membrane within W/O droplets.

Why are water-in-oil droplets used in this reconstitution approach?

W/O emulsion droplets provide confined geometry that mimics the intracellular environment and supports dynamic interaction between actomyosin and lipid membranes.

Sep 29, 2026

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