, 2007). We next induced expression of these constructs in established cocultures that had myelinated for 6–8 weeks beforehand.

Myelination is largely complete in such cocultures as evidenced by Nutlin-3 nmr the stable numbers of nodes and heminodes even after an additional 3 weeks of coculture (Figure S6). In contrast to forming nodes, only WT NF186 and ICAM/NF were targeted appropriately to mature nodes, i.e., those flanked on both sides by paranodes, whereas NF/ICAM and NF186ΔABD were not targeted (Figures 6D and 6F). Also in contrast to young cocultures, ICAM/NF was expressed at much lower levels along the internodes of most myelinated fibers. Extraction of such cultures with Triton X-100 prior to fixation eliminated residual ICAM/NF from the internode, but not the nodes, where it remained colocalized with ankyrin G (Figure S5C). These latter results support the notion that binding to ankyrin

G promotes stable expression of NF186 at mature nodes. Interestingly, ICAM/NF is not targeted to heminodes in these cultures (Figures 6F and S5B) despite the enrichment of ankyrin G at these sites. These results indicate that the cytoplasmic domain of NF186 is sufficient to mediate targeting Protease Inhibitor Library cell assay to mature nodes, whereas the ectodomain is also required for targeting to heminodes. Taken together, these results indicate that NF186 is targeted to nascent nodes and mature nodes by distinct mechanisms: ectodomain sequences are necessary and sufficient for initial accumulation at heminodes and newly formed nodes, whereas intracellular sequences isometheptene are necessary and sufficient for targeting and stable accumulation at mature nodes. They also suggest that the paranodes modify the mechanisms by which NF186 accumulates at nodes. To extend these findings, we generated transgenic mice expressing WT NF186, NF/ICAM, and ICAM/NF under

the control of the neuron-specific Thy-1.2 promoter ( Aigner et al., 1995 and Feng et al., 2000). Each construct was tagged with EGFP at the C terminus. We generated several founders for each construct and analyzed expression by staining for GFP. The overall pattern of expression was similar for different founders that expressed the same construct. Representative images for one founder line for each construct are shown at different postnatal ages in Figure 7A. NF186 and NF/ICAM were concentrated at nodes (arrowheads, Figures 7A and 7B) and heminodes (arrows, Figure 7B) by postnatal day 3 (P3), the earliest time point analyzed. While WT NF186 persisted at nodes at all time points examined, NF/ICAM was greatly reduced at nodes at P14 and largely absent in the adult, indicating that stable nodal expression of NF186 requires cytoplasmic interactions, presumably with ankyrin G.