Live imaging confirmed that transport of labeled vesicles was blocked by BFA see more (data not shown). As Schwann cells do not myelinate in the
presence of BFA (data not shown), we established microfluidic chambers, which allowed neurons to be treated separately from Schwann cells. In these cultures, neuronal cell bodies and their distal neurites are grown in separate compartments, connected by processes that extend through microgrooves (Taylor et al., 2005); Schwann cells were added to the neurite compartment and maintained under myelinating conditions (Figure 2D). The compartment containing the cell bodies was treated either with vehicle control (DMSO) or with BFA continuously, beginning with the onset of myelination. Cultures were then fixed, and domain markers were analyzed in the Schwann cell-neurite compartment. As shown in Figure 2E, and quantified in Figure 2F, treatment with BFA blocked accumulation of sodium channels and ankyrin G, but not that of adhesion molecules (i.e., NF186 and Caspr). Like the transected Nmnat1-protected axons, the effects of BFA were most pronounced Selleckchem 5 FU on ankyrin G accumulation; occasional sodium channel clusters devoid of ankyrin G were observed (Figure S2C). These findings strongly support the notion that ion channels and their
cytoskeletal scaffold require transport from the soma, whereas adhesion molecules (i.e., NF186, NrCAM) accumulate at the node from local (i.e., transport-independent) stores. To investigate whether these distinct routes of accumulation correlate to differences in the planar mobility of these components, we analyzed the diffusion of each of these proteins in the axon membrane. We first nucleofected neurons with GFP-tagged NF186 (Dzhashiashvili et al., 2007), NrCAM, NaV1.2, KCNQ3, and ankyrin G constructs. We analyzed NaV1.2, which is expressed transiently at forming PNS nodes (Boiko et al., 2001 and Rios et al.,
2003) and is more readily expressed after transfection of neurons than NaV1.6 (Lee and Goldin, 2009). Each of these constructs was diffusely expressed along unmyelinated axons and localized appropriately to heminodes (Figure 3A) and nodes (insets, Figure 3A) MycoClean Mycoplasma Removal Kit of Ranvier with myelination. We next measured the mobility of these nodal components in individual, unensheathed neurites by FRAP (fluorescence recovery after photobleaching) (Snapp et al., 2003). Representative results from photobleaching experiments are shown in Figure 3B; intensity measurements (Figure 3C) and a summary of the calculated mobilities (Figure 3D) are also shown. In general, NF186 and NrCAM were uniformly mobile with diffusion coefficients for NF186 of 0.338 ± 0.022 μm2/s (mean ± SEM, n = 12) and for NrCAM of 0.198 ± 0.016 μm2/s (n = 6); in both cases, the fluorescence recovery was nearly complete, indicating that the population is fully mobile. In contrast, the mobility of ion channels NaV1.