This selective recruitment of CAMs, this website but not of channel and cytoskeletal proteins,

to the heminodes of transected axons does not result from differences in the turnover or abundance of these proteins following axonal transection. Thus, western blotting analysis (Figures S1B and S1C) demonstrated comparable turnover of the nodal components NF186, NaChs, and ankyrin G at 7 days following axotomy, the approximate time the nodes were analyzed. These components were also detectably expressed in neuron-only cultures after axotomy based on immunostaining (data not shown). These findings indicate that differential recruitment to the nodes is not the result of differential availability. Rather, they suggest that CAMs and channels are directed to the node from different pools of proteins. Two potential Bortezomib concentration sources

of axonal proteins may contribute to node assembly: (1) surface proteins that redistribute to this site, and/or (2) proteins in intracellular vesicles that are transported there. To distinguish between these possible sources further, we characterized the effect of axotomy on vesicle transport by imaging Nmnat1-labeled (Figure 2A) or NF186-GFP labeled vesicles (data not shown) with time-lapse microscopy. Vesicle transport largely ceased 8 hr after axotomy (Figure 2A; Move S1. Active Trafficking of Intracellular Vesicles in Nmnat1-Positive Axons prior to Transection, Related to Figure 2A and Movie S2. Markedly Reduced Trafficking of Intracellular Vesicles in Nmnat1-Positive Axons after Transection, Related to Figure 2A) even though there were no obvious changes in the organization of microtubules or neurofilament

after transection (Figure 2B). Similar results were observed Non-specific serine/threonine protein kinase in myelinating cocultures (data not shown). As nodes did not form until 3 or 4 days after axotomy, and paranodes later still, these results strongly suggest that adhesion molecules detected at heminodes (NF186, NrCAM) and paranodes (Caspr) in the transected axons did not accumulate via transport, in contrast to ion channels and cytoskeletal proteins. To directly examine the role of vesicular transport during node assembly, and whether newly synthesized proteins from the soma contribute, we treated neurons with brefeldin A (BFA); this treatment results in mixing of the ER and Golgi compartments, blocking anterograde, vesicular trafficking (Klausner et al., 1992). We first confirmed that BFA blocks transport of newly synthesized proteins into the axons over extended time periods. We inducibly expressed NF186, tagged with GFP at its C terminus (Dzhashiashvili et al., 2007), in neurons under the control of the doxycycline-inducible lentiviral vector pSLIK (Shin et al., 2006).

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