Myelinated fibers are organized into anatomically and physiologically distinct longitudinal domains - the internode, the paranodes and the node of Ranvier. This longitudinal organization results from complex interactions between the axon and myelinating glial cells that are essential for the rapid and efficient conduction of impulses via saltatory conduction. Voltage gated Na+ and delayed rectifier K+ channels are concentrated in the nodal and juxtaparanodal regions respectively, with specialized septate-like junctions interposed in-between. The precise function of these junctions is not known. They are likely to provide a site of adhesion between these cells and a partial barrier to ion flow; they may also regulate the distribution of voltage gated channels nd mediate reciprocal signaling between axons and glia. We have recently demonstrated that Caspr is a major axonal constituent of these junctions, the first such component to be identified. Critical unanswered questions include what regulates the formation of these junctions, what are their functions, and what other molecules are associated with Caspr in the paranodes? To address these question we will: i) Determine the sequence of events during formation of the node and the paranodes in a myelinating co- culture system and determine whether node formation is a prerequisite of Caspr localization, ii) Analyze the function of Caspr as an adhesion molecule and its role in the formation of the paranodal junctions. To this end, we will generate and purify a Caspr-Fc chimeric protein for binding studies with Schwann cells and oligodendrocytes and for perturbation studies of paranodal junction formation in vitro and iii) isolate novel proteins that are associated with Caspr in the paranodal junctions and characterize its interactions with several known proteins by isolating non-ionic detergent complexes enriched in Caspr and associated proteins. We will also use the Caspr-Fc to characterize its presumptive glial receptor by cross-linking and affinity purification techniques and continue to analyze proteins that may interact with the cytoplasmic domain of Caspr including PI 3-kinase.
