Actin-based cell motility plays crucial role throughout the lifetime of an organism. While the dendritic nucleation model explains the initiation and organization of the actin network in lamellipodia, two questions need to be answered.

In this study, I reconstructed cellular motility in vitro to investigate how actin filaments are organized to coordinate elongation and attachment to leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel beads or thin glass nanofibers coated with N-WASP WCA domains. During sustained motility, physiological concentrations of Mg²⁺ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg²⁺ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, filament capping, or actin shell formation. Addition of other types of crosslinkers restored both comet tail attachment and particle motility. We propose a model in which polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.

I lowered actin, profilin, Arp2/3, and CP concentrations to address the generation of actin filament orientation during the initiation of motility. In the absence of CP, Arp2/3 nucleates barbed ends that grow away from the nanofiber surface and branches remain stably attached to nanofiber. CP addition causes shedding of short branches and barbed end capture by the nanofiber. Barbed end retention by nanofibers is coupled with capping, indicating that WWCA and CP bind simultaneously to barbed ends. In pull-down assays, saturating CP addition only blocks WWCA binding to barbed end by half. Labeled WWCA bound to barbed ends with an affinity of 14 pM and unlabeled WWCA with an affinity of 75 pM. CP addition increased WWCA binding slightly at low CP concentrations and decreased WWCA binding to 50% at high CP concentrations. Molecular models of CP and WH2 domains bound respectively to the terminal and penultimate actin subunit showed no overlap and that CP orientation might blocks WWCA dissociation from the penultimate subunit. Simultaneous binding of CP and WWCA to barbed ends is essential to the establishment of filament orientation at the leading edge.