Date of Award
Doctor of Philosophy (PhD)
The cerebellum is essential for learning coordinated movements, balance, eye movements, and aspects of sensory cognition. The remarkably uniform array of the principal neurons of the cerebellar circuitry, granule cells and Purkinje cells, develops by glial-guided migrations that position these cell types in specific neuronal layers. Classical EM studies and real time imaging of the migration of granule neurons along glial fibers established the mode of movement of migrating neurons, including the formation of an interstitial junction beneath the cell soma. Biochemical and molecular biological experiments demonstrated that neuronal protein astrotactin (ASTN1) functions as a neuron-glial ligand during migration. This thesis presents the cloning and biochemistry of a second member of the Astn gene family, Astn2. Although the peptide sequence of ASTN1 and ASTN2 is highly conserved, we demonstrate that different domains of ASTN1 and ASTN2 are exposed on the cell surface. Immunoprecipitation experiments show that ASTN1 associates with ASTN2 and flow cytometry analysis reveals that coexpression with ASTN2 alters the cell surface localization of ASTN1. Thus, implying that ASTN1 forms a complex with ASTN2. Recent evidence indicates the polarity complex mPar6 α regulates forward movement of the neuron along the glial fiber by coordinating the forward translocation of the centrosome and nucleus. Studies presented in this thesis provide evidence that the junction beneath the cell soma is released by endocytosis of the ASTN1:ASTN2 complex. Real time imaging of labeled ASTN1 shows a flow of protein to the front of the cell and base of the leading process as the neuron moves along the glial guide. Double labeling with fluorescently tagged Clathrin light chain suggests that the endocytosis of ASTN1 and ASTN2 occurs by a clathrin-related mechanism. Based on these results we present a model whereby the endocytosis of the neuron-glial ligands, ASTN1 and ASTN2, releases adhesion to the glial guide, after which the neuronal soma moves along the glial fiber until a new adhesion junction forms. Thus, studies on ASTN2 in this thesis show a mechanism for the termination of neuron-glial adhesion required for neuronal movement along the glial fiber
Wilson, Perrin Megan, "Characterization and Functional Analysis of Astrotactin 2 Reveals a Role for Endocytosis in Glial-guided Neuronal Migration" (2007). Student Theses and Dissertations. 186.