In contrast, ICAM/NF was only modestly enriched at nodes at P3 (Figures 7A and 7B) but progressively BMS-354825 cost concentrated at nodes over time into adulthood. It was expressed at

high levels along the internode at P3 and P14, consistent with a requirement for the NF186 ectodomain in internodal clearance (Dzhashiashvili et al., 2007); its expression along the internode of adult nerves was limited. Notably, while ICAM/NF was enriched at many nodes, it was not enriched at heminodes at P3 (Figure 7B), a time when heminodes are still abundant; this lack of heminode expression contrasts to that of WT NF186 and NF/ICAM. The differences in the localizations of these constructs at P3 and in the adult are quantified in Figure 7C. To assess the association of these NF constructs with the cytoskeleton, we extracted sciatic nerves with Triton X-100, then fixed and stained for GFP (Figure 7D) similar to our analysis of the cocultures. As expected, WT NF186 was detergent resistant, whereas NF/ICAM was extracted from nodes and heminodes at P3. In contrast, ICAM/NF was extracted from nodes and the axon at P3, but not from nodes at P14 (Figure 7D). These results Galunisertib price are quantified in Figure 7E and suggest that while

ICAM/NF accumulates at the node by P3, it is not yet stably associated with the cytoskeleton. In this study, we have examined the sources of axonal proteins that contribute to node formation and maintenance. We demonstrate that the adhesion molecules NF186 and NrCAM are recruited to nascent nodes from diffusible, preexisting surface pools, whereas ion channels and cytoskeletal components, which are mostly immobile, rely on transport. We have characterized the targeting of

NF186 further and show that it redistributes to nascent nodes via ectodomain interactions, whereas it is replenished at mature nodes by direct targeting that relies on intracellular interactions. These findings, summarized schematically in Figure 8, and their implications for the mechanisms of node assembly and maintenance are discussed below. A key finding is that adhesion molecules that Sodium butyrate nucleate the domains of myelinated axons are likely to redistribute from existing pools on the axon surface. Thus, NrCAM and NF186 accumulated at heminodes, and Caspr at paranodes, during myelination of transected Nmnat1-protected axons or in BFA-treated microfluidic cocultures indicating that they are derived from local sources and do not require transport from the soma. The accumulation of a surface biotinylated AviTag-NF186 construct at newly formed nodes and heminodes provides direct evidence that it redistributes from a surface pool (Figure 4C). While a formal possibility, transcytosis of surface proteins (Wisco et al., 2003), i.e.