Reconstituting Myosin-I-Mediated Branched Actin Assembly in a Bead-Based Actin Comet-Tail Motility Assay.

Branched actin polymerization mediated by the actin-related protein 2/3 (Arp2/3) complex provides the primary pushing forces for a variety of cellular processes, including cell migration, endocytosis, and phagocytosis. Myosin-I motors, which frequently colocalize with branched actin networks at the cell leading edge, have also been shown to participate in these processes and are thought to regulate actin network organization and mechanical output. However, the molecular mechanisms by which myosin-I interacts with the Arp2/3 complex to modulate branched actin assembly and force generation remain largely unknown. Here, we describe a highly tunable in vitro actin comet-tail bead motility assay that reconstitutes the interplay among myosin-I, actin, and the Arp2/3 complex at the cell leading edge on the surface of micron-sized beads. This method is adapted from well-established actin comet-tail assays by co-immobilizing myosin-I with nucleation-promoting factors (NPFs) on bead surfaces, thereby creating a membrane-like actin assembly interface. The assay enables visualization and quantitative analysis of actin network assembly, network density, bead motility, and growth efficiency. It also provides indirect, qualitative readouts of myosin-I-mediated force enhancement in branched actin networks. This protocol includes bead functionalization, reaction assembly, fluorescence imaging, quantitative image analysis, and troubleshooting strategies providing a reproducible platform for studying myosin-I-regulated actin assembly at membrane-like interfaces.