The axon initial segment (AIS) and nodes of Ranvier are sites of action potential generation and regeneration, respectively and are thus critical for the proper function of the nervous system. Their key roles in electrogenesis results from the striking enrichment of a macromolecular complex of voltage-gated sodium (NaV) and potassium (KCNQ) channels, cell adhesion molecules (NF186, NrCAM), and a cytoskeletal scaffold of ankyrin G (AnkG) and beta-IV (βIV) spectrin. Prior to myelination, nodal components are diffusely distributed along axons consistent with their continuous conduction of action potentials. With myelination, the axon reorganizes into discrete domains, culminating in node assembly, thus enabling saltatory conduction. A key question is what drives this reorganization? We have found two complementary mechanisms broadly contribute: i) recruitment signals that target and stabilize this complex at nodes and ii) active clearance that removes nodal proteins from everywhere else along the axon. Here, we examine the contribution of both mechanisms to node formation. Recruitment signals at PNS and CNS nodes culminate in assembly of an AnkG/βIV spectrin cytoskeleton scaffold to which all other nodal components bind. This scaffold is further tethered to and likely stabilized by regularly spaced, sub-membranous actin rings at both the AIS and nodes. We recently reported actin rings at the AIS and nodes are specifically modified by contractile myosin II. In particular, phosphorylated myosin light chain (pMLC) - the regulatory subunit that activates the contractile function of myosin II - is enriched at and an early marker of the AIS and nodes. Strategies that increase or decrease pMLC levels/myosin II activity, drive AIS assembly and disassembly, respectively. These results implicate contractile NMII as a novel regulator of the AIS and suggest a conserved role at nodes, a notion strongly supported by MLC knockdown studies. We have also found that just prior to myelination, glial cells drive clearance of nodal components from the internode by clathrin-mediated endocytosis (CME). Our results suggest cleared proteins are not linked to the cytoskeleton and can therefore be clustered for endocytosis. In agreement expression of a mutant NF186 construct engineered to bind to the internodal cytoskeleton, is not endocytosed but rather is persistently expressed along the axon. Strikingly, this construct delays myelination. This latter finding suggests clearance has dual roles in sculpting the node and preparing the axon for myelination thereby coordinating assembly of the node of Ranvier with myelination of axons. Here, we test key aspects of this model, including the role of MLC/myosin II in node assembly/stability and actin ring integrity by knockdown and knockout strategies. We will also examine the mechanisms and consequences of this glia-driven clearance of axonal proteins, including modeling defective CME in mice to broadly perturb the axon surface pr...