Development of the axonal βII-spectrin periodic skeleton requires active cytoskeletal remodelling

The axonal membrane-associated periodic skeleton (MPS), consisting of F-actin rings crosslinked by spectrin heterotetramers, is ubiquitous and critical for neuronal function and homoeostasis. However, the initiation and early development of the axonal MPS are poorly understood. Using superresolution imaging, we show that βII-spectrin is recruited early to the axonal cortex, followed by progressive establishment of long-range periodic order. Microtubule dynamics are essential for MPS formation in the early stages, but transition to a passive stabilising role in mature axons. We show that the early subplasmalemmal recruitment of βII-spectrin is dependent on cortical actin but not on actomyosin contractility, and active nucleation of F-actin is required in early development but is dispensable for the mature MPS. Using a βII-spectrin knockout model, we demonstrate that the actin-binding and lipid-interacting domains of βII-spectrin are critical for its subplasmalemmal confinement and, subsequently, MPS maturation. These findings highlight stage-specific cytoskeletal remodelling underlying MPS development and advance our understanding of axonal subcellular architecture.