The overall goal of this research is to use an in vitro model system, developed in our laboratory, to study the molecular mechanism of glial-guided neuronal migration in the developing brain. The proposed research will purify and characterize a novel glycoprotein, ASTROTACTIN, antibodies against which block neuron-glial interactions in the microculture system. Monoclonal and mono-specific polyclonal anti-astrotactin antibodies, selected with in vitro functional assays, will be used to purify astrotactin, to select cDNA clones from a mouse lambda gtll expression library and to isolate fusion proteins from immunopositive clones. High resolution video microscopy will then be combined with computer-assisted image processing to visualize the effects of Fab fragments of mono-specific astrotactin antibodies on the interaction of the migrating neuron and its leading process with the glial fiber. Biochemical studies on astrotactin and its proteolytic cleavage products will be used to analyze the size, shape and functional domains of the molecule. This information will be combined with in vitro assays of the effects of astrotactin peptides, fusion peptides and monoclonal antibodies to define the regions of astrotactin that regulate neuron-glial interactions. We will then use immunocytochemical and molecular biological methods to study the expression of astrotactin in the developing C57BL/6J mouse brain and in the weaver mouse mutant mouse, because we have found that astrotactin is missing or defective in the medial aspect of the weaver cerebellum. Finally, we will raise anti-idiotypic antibodies against anti-astrotactin antibodies and use affinity chromatography to attempt to purify a receptor for astrotactin from glial cells.
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