is shown on the right. from the nodes, the formation of binary nodes, and dysregulation of nodal gap length. Therefore, these mice exhibit neurological abnormalities and slower nerve conduction. Disintegration of the nodes occurred in an orderly manner, starting with the disappearance of neurofascin 186, followed by the loss of Na+ channels and ankyrin G, Adam30 and then IV spectrin, a sequence that reflects the assembly of nodes during development. Finally, the absence of gliomedin and NrCAM led to the invasion of the outermost D-Cycloserine layer of the Schwann cell membrane beyond the nodal area and the formation of paranodal-like junctions at the nodal gap. Our results reveal that axon-glial contact mediated by gliomedin, NrCAM, and NF186 not only plays a role in Na+ channel clustering during development, but also contributes to the long-term maintenance of Na+ channels at nodes of Ranvier. test. Results Mice lacking both gliomedin and NrCAM exhibit neurological abnormalities and slower nerve conduction Because both gliomedin and NrCAM interact directly with NF186 (Eshed et al., 2005) and could thus potentially provide redundant functions in mature nodes, we generated mutant mice lacking both genes (Fig. 1= 8). 7 mice per genotype; 0.001. 0.0001. Scale bar, 10 m. In contrast to wild-type or the single mutants, Na+ channels in double = 3C5 animals per age per genotype (300 nodes per group), * 0.001. = 3C5 animals per genotype per age (counted at least 300 sites per group), * 0.005. Scale bars, 5 m. Open in a separate window Figure 4. Molecular composition of the nodes in the absence of gliomedin and NrCAM. Immunolabeling of teased sciatic nerve fibers isolated from adult wild-type (and insets). Merged images are found on the right of each panel. Note the segregation of NF186, ankyrin G, and IV-spectrin, but not of pERM, with nodal Na+ channels. Scale bar, 5 m. The observation that axonodal components disintegrate and form two separate bands prompted us to measure the length of the nodal gap using longitudinal sections of adult sciatic nerves from all genotypes (Fig. 3(showing a paranode-type junction at which the apposed membranes are separated by only 2C4 nm (arrowheads). The junctional gap at these sites is irregularly dense, but discrete transverse bands cannot be resolved because of the angle of the section. is shown on the right. Asterisks mark the location D-Cycloserine of Caspr within the nodes, which is flanked by binary clusters of Na+ channels and IV spectrin. = 3 animals per genotype per age (300 sites counted per group), 0.005. em E /em , Schematic summary of node disassembly in the absence of gliomedin and NrCAM ( em a /em C em d /em ). Loss of NF186 precedes that of Na+ channels and AnkG, which occurs before the loss of IV spectrin from the nodes. Discussion Nodes in the PNS are assembled by two cooperating mechanisms: clustering of nodal components at heminodes and their restriction to the nodal gap by the adjacent paranodal junction (Feinberg et al., 2010). Initial clustering of Na+ channels occurs at heminodes and is formed at the edge of myelin segments. This process is mediated by binding of Schwann cell-derived gliomedin and NrCAM to their axonal receptor NF186 (Eshed et al., 2005; Feinberg et al., 2010). Gliomedin is localized to the Schwann cell microvilli by glial NrCAM (Feinberg et al., 2010) and by its association with the surrounding nodal D-Cycloserine ECM (Eshed et al., 2007). The concentration of gliomedin by NrCAM and the nodal ECM forms high-avidity complexes that trap NF186 and cluster it on the underlying axolemma (Zhang et al., 2012). In the absence of a functioning heminodal clustering mechanism (e.g., in mice lacking gliomedin or NrCAM; Feinberg et al., 2010), node assembly is driven solely by the flanking paranodal junctions, which serve as diffusion barriers that limit the lateral movement of membrane proteins (Rosenbluth, 2009). In agreement, we found that nodes did form in the absence of both gliomedin and NrCAM, further supporting a primary role for the paranodal-junction-dependent mechanism in the assembly of PNS nodes. Nevertheless, the delay in the accumulation of Na+ channels in mature nodes in the double em gldn /em ?/?/ em nrcam /em ?/? mutant, together with the normal appearance of nodes in paranodal mutant mice (Bhat et al., D-Cycloserine 2001; Boyle et al., 2001; Gollan et al., 2003), indicate that, in the.