Fig 2: Schematic depicting the peripheral nerve and node of Ranvier. (a) In the peripheral nervous system (PNS) nerves extend from their cell body to their target muscle or organ where they terminate, in this example at neuromuscular junctions (NMJ) on the muscle fibre. Nerves are wrapped in myelin formed by Schwann cells (oligodendrocytes in the CNS). Nodes of Ranvier (NoR) are formed by gaps in the Schwann cells and propagate the action potential. NoR consist of three major domains: the nodal gap is flanked by the paranodes (PN) where the myelin loops attach to the axon. The paranodes in turn are flanked by the juxtaparanodes (JPN). (b) Here, we depict an enlarged representation of a heminode. In each nodal domain there is a special arrangement of axonal and glial proteins that promotes the high-density clustering of ion channels. Here we depict what is currently understood about glycolipid lipid raft distribution in the nodal membranes and focus on their relationship to key proteins. As such, we recognise that for simplicity we have omitted a number of the proteins within the axons, glia and extracellular matrix that form the NoR. Paranodal axonal proteins Caspr and Contactin are likely highly associated with a-series gangliosides and are disturbed in their absence. Interaction with their glial binding partner neurofascin 155 (NF155), forms the paranodal axo-glial junction. NF155 and myelin associated glycoprotein (MAG) are likely tethered by sulphatide rafts in the paranodal glial membrane. GD1a and GT1b are represented in the axonal paranodal ganglioside rafts as they are the major ligands of MAG, however, in reality the rafts could contain all complex gangliosides. Under protein/lipid deficient or pathological conditions, lengthening of Nav channel domains are indicated by the pink arrow, and invasion of juxtaparanodal Kv channels into the paranode, suggestive of breakdown of the axo-glial junction at the paranodal/juxtaparanodal border, by the green arrow

Fig 3: (a) Peripheral nerve nodal organisation is unchanged among mice with selective neural membrane ganglioside expression. Sciatic nerve from 4 to 6 week old wild-type, Neuronal (GalNAc-T -/--Tg(neuronal)), Glial (GalNAc-T -/--Tg(glial)), Neuronal x Glial, and GalNAc-T -/- mice were analysed (n = 3/ genotype). There were no significant differences among the genotypes in the gap between juxtaparnodal Kv1.1 domains, pan neurofascin (pan-NFasc, binds both neurofascin 155 and 186) domain length, Caspr domain length or Nav channel cluster length (one-way ANOVA, p > 0.05). (b) Illustrative merged images show immunostaining for: top panel Kv1.1 (orange) and Caspr (magenta); bottom panel pan-NFasc (orange) and Nav channels (magenta). Caspr immunostaining was located at the paranodes and flanked by juxtaparanodal Kv1.1 in all nerves. Distinct Nav channel clusters were flanked by pan-Nfasc domains (presumed glial NF155). Paranodal invasion was occasionally observed in Glial and GalNAc-T -/- mice (indicated by arrowhead). Scale bar = 5 µm

Fig 4: The relationship between Nav1.2 and Nav1.6 localizations and the myelination status of pyramidal neurons. A, B, Double immunostaining of V5-labeled Nav1.2 with MBP (red) in unmyelinated neuron (A) and partially myelinated neuron (B). Scale bar, 10 µm. C, Double immunostaining of V5-labeled Nav1.6 with MBP (left) and Caspr (right). Scale bar, 10 µm. D, The percentage of unmyelinated, partially myelinated, and myelinated neurons in Nav1.2- or Nav1.6-positive knock-in neurons. The number of cells analyzed, Nav1.2, 60; Nav1.6, 97. Movies 2, 3, 4 show 3D image details.