Axon initial segments (AISs) and nodes of Ranvier are sites of action potential generation and propagation, respectively. Both domains are enriched in sodium channels complexed with adhesion molecules (neurofascin [NF] 186 and NrCAM) and cytoskeletal proteins (ankyrin G and βIV spectrin). We show that the AIS and peripheral nervous system (PNS) nodes both require ankyrin G but assemble by distinct mechanisms. The AIS is intrinsically specified; it forms independent of NF186, which is targeted to this site via intracellular interactions that require ankyrin G. In contrast, NF186 is targeted to the node, and independently cleared from the internode, by interactions of its ectodomain with myelinating Schwann cells. NF186 is critical for and initiates PNS node assembly by recruiting ankyrin G, which is required for the localization of sodium channels and the entire nodal complex. Thus, initial segments assemble from the inside out driven by the intrinsic accumulation of ankyrin G, whereas PNS nodes assemble from the outside in, specified by Schwann cells, which direct the NF186-dependent recruitment of ankyrin G.

The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity.

Summary We have investigated the source(s) and targeting of components to PNS nodes of Ranvier. We show adhesion molecules are diffusible within the axon membrane and accumulate at forming nodes from local axonal sources. In contrast, ion channels and cytoskeletal components exhibit limited planar mobility and require transport to the node. We have characterized further targeting of NF186, an axonal adhesion molecule, which pioneers node formation. NF186 redistributes to nascent nodes from a mobile, surface pool. This initial accumulation, and clearance from the internode require extracellular interactions whereas subsequent targeting to mature nodes, i.e. those flanked by paranodal junctions, requires intracellular interactions. After incorporation into the node, NF186 is immobile, stable, and promotes node integrity. Thus nodes assemble from two sources: adhesion molecules, which initiate assembly, accumulate by diffusion trapping via interactions with Schwann cells whereas ion channels and cytoskeletal components accumulate via subsequent transport. In mature nodes, components turnover slowly and are replenished via transport.

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