Myosin-I enhances the force generation of Arp2/3-mediated branched actin assembly

Actin and myosin are molecular machineries that convert free energy released from ATP hydrolysis into mechanical force. Polymerizing actin filaments and myosin motor activity generate forces that power a variety of cellular processes. Myosin-I proteins, commonly found alongside branched actin networks stimulated by Arp2/3 complex, play a key role in force generation for membrane deformation and cell protrusion. Yet, the molecular mechanism of how myosin-I coordinates with branched actin assembly to generate force remains largely unknown. To investigate the role of myosin-Is in branched actin assembly, we reconstituted an in vitro actin-based bead motility system, where branched actin networks were nucleated by Arp2/3 complex on the surface of micron-sized beads coated with Arp2/3 activating factors. Actin filaments first formed a symmetric shell around the bead, which transitioned into a polarized comet tail that propelled the bead forward after symmetry breaking. We site-specifically coupled various densities of myosin-Is to the bead surface and assessed their effects on actin polymerization, network architecture, and symmetry breaking. We also developed filament-level dendritic network simulations to further mimic the effects of myosin-I on actin networks. Our findings suggest that myosin-I enhances the force generation of Arp2/3-mediated branched actin assembly by regulating the actin assembly kinetics and network architecture through its force-generating power stroke.