Abstract
The cell adhesion molecules (CAMs) of the immunoglobulin superfamily (Ig-CAMs) play a crucial role in the organization of the node of Ranvier in myelinated axons. In the peripheral nervous system, Gliomedin (Gldn) secreted by Schwann cell microvilli binds NgCAM-related CAM (NrCAM) and Neurofascin-186 (NF186) and direct the nodal clustering of voltage-gated sodium channels (Nav). NF186 is the single axonal Gldn partner to ensure Nav clustering at nodes, whereas NrCAM is only required in glial cells (Feinberg, K., Eshed-Eisenbach, Y., Frechter, S., Amor, V., Salomon, D., Sabanay, H., Dupree, J. L., Grumet, M., Brophy, P. J., Shrager, P., and Peles, E. (2010) Neuron 65, 490-502). The olfactomedin domain of Gldn is implicated in the interaction with nodal Ig-CAMs. However, the interacting modules of NrCAM or NF186 involved in Gldn association are unknown. Here, we report that fibronectin type III-like (FnIII) domains of both Ig-CAMs mediate their interaction with Gldn in pulldown and cell binding assays. Using surface plasmon resonance assays, we determined that NrCAM and NF186 display similar affinity constant for their association with Gldn (K(D) of 0.9 and 5.7 nm, respectively). We characterized the FnIII domains 1 and 2 of NF186 as interacting modules that ensure association with Gldn. We found that the soluble FnIII domains of NF186 (FnIII-Fc) bind on Schwann cells and inhibit Gldn and Nav clustering at heminodes, the precursors of mature nodes in myelinating cultures. Our study reveals the unexpected importance of FnIII domains of Ig-CAMs in the organization of nodes of Ranvier in peripheral axons. Thus, NF186 utilizes distinct modules to organize the multimeric nodal complex.
Highlights
Neurofascin is a cell adhesion molecule that mediates axo-glial interactions at the nodes of Ranvier
fibronectin type III-like (FnIII) Domains of NgCAM-related CAM (NrCAM) Are Required for Interaction with Gldn—Previous studies demonstrated that the olfactomedin domain of Gldn mediates its interaction with NF and NrCAM [7, 8]
To dissect the basis for Ig-cell adhesion molecules (CAMs)/Gldn association further, we investigated whether the Ig or FnIII domains of NrCAM and NF may be critical for these interactions
Summary
Neurofascin is a cell adhesion molecule that mediates axo-glial interactions at the nodes of Ranvier. Two cell adhesion molecules of the immunoglobulin superfamily (Ig-CAMs), the axonal 186-kDa isoform of Neurofascin (NF186) and NrCAM, first accumulate at high density and precede the Nav clustering at heminodes, which will fuse to form the mature node [5, 6]. Several studies demonstrated the distinct roles of glial NF155 and neuronal NF186 in the formation of the paranodes and nodes, respectively [13, 14, 17, 18] These two isoforms contain distinct domains due to alternative splicing: i.e. NF186 encompasses a juxta-membrane mucin-like domain and NF155 an additional Fibronectin type III-like (FnIII) domain. Functional analysis using myelinating cultures of DRG neurons indicates that treatment with soluble FnIII domains of NF186 prevents the clustering of Gldn at heminodes, as observed with fulllength NF186
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