Distinct Arp2/3 isocomplexes drive axonal actin ring assembly and integrity

The actin-related protein 2/3 (Arp2/3) complex is a central nucleator of branched actin filaments, essential for numerous cellular processes. Using superresolution imaging, we demonstrate that Arp2/3 is a component of the membrane periodic skeleton (MPS)-a highly ordered actin ring-spectrin lattice in the submembrane axonal cytoskeleton. While generally viewed as a single entity, functional specialization of the Arp2/3 complex arises from isoform diversity of three of its seven subunits. Moreover, isoform-specific perturbations revealed that distinct Arp2/3 isocomplexes orchestrate successive stages of MPS actin ring development. While Arp2/3 isocomplexes containing ARPC1A and ARPC5 are essential for actin ring assembly during early MPS formation, those with ARPC1B and ARPC5L are key to maintaining actin rings in mature axons. These findings demonstrate that beyond linear actin, branched actin filaments contribute to the MPS nanoarchitecture and uncover a switch in Arp2/3 isocomplex composition that is required to ensure actin ring assembly and stability.