Our laboratory has made the first identification of an immunoreactive, structurally related analogue of red blood cell spectrin in various nonerythroid cells. Recently, we have identified and purified an immunoreactive analogue of rbc spectrin from a membrane fraction of mouse brain. The purified brain spectrin-like molecular is a large asymmetric 972,000 MW, 10.5S, (AlphaBeta)2 tetramer, consisting of 240,000 dalton and 235,000 dalton subunits. While brain spectrin-like protein and erythrocyte spectrin share common antigenic sites, morphology, subunit composition, and functional regions, two dimensional tryptic peptide analysis indicates only limited homology between the mouse brain and erythrocyte proteins. Antibodies against native brain and rbc spectrin have been used to study adult mouse brain by immunofluorescence, and neuronal perikarya and cell processes are particularly enriched in spectrin. In studies described in this proposal we examine the role of spectrin-like proteins in the developing mouse brain. Intensive immunobiochemical and immunofluorescent investigations of mice of ages 15 days prenatal, newborn, and 10, 21, 30, and 90 days postnatal will allow us to focus on active periods of neuronal and glial cell proliferation, migration, and differentiation. Antibodies to 240K rbc, 220K rbc, 240K brain and 235K brain spectrin subunits will be used in indirect immunofluorescent studies that will localize spectrin in brain tissue as well as within neural cells; structural correlates using phase microscopy, silver staining, and gallocyanin stained sections will be included. Immunoelectron microscopy with ferritin-labeled antibodies will be undertaken for ultrastructural localization of spectrin subunits. The type and quantity of spectrin-like subunits during neuro-ontogeny will be determined with immunoprecipitation and immunoautoradiographic techniques utilizing our antibodies to the spectrin subunits. Immunofluorescent images will thereby be correlated with the identification of the subunit being visualized. In addition, one and two dimensional peptide mapping of radioiodinated polypeptides will be used to ascertain structural changes in the brain spectrin subunits during development. The studies proposed will be the first inquiry into the functional significance of spectrin-like proteins in the developing nervous system and reveal the roles of spectrin in neural cells and tissues. This investigation is part of an ongoing program in cellular and molecular neurobiology which seeks to understand the processes of normal and abnormal brain development.
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