In brain, the composition of microtubule protein (tubulin and its associated proteins) is both complex and heterogeneous. The heterogeneous composition of microtubules is manifested not only by changes during development, but also by differences in cellular, subcellular and regional distribution. The significance of this heterogeneity is not understood, but it has been proposed that variations in microtubule protein composition reflect the multiple functions which microtubules appear to perform. The objective of this proposal is to identify specific microtubule populations by their microtubule-associated protein (MAP) content. Specifically, two MAPs, MAP2 and tau will be studied. Microdissection methods, subcellular fractionation procedures and in vitro microtubule assembly will be used to isolate microtubule proteins in specific tissue samples from adult and developing brain. These fractions will be analyzed by quantitative enzyme-linked immunosorbant assays (ELISAs) and gel scanning to determine the ratios of MAP2 and tau to tubulin, and to identify the structural forms (electrophoretic variants) of MAP2 and tau present. The entire complement of polypeptides which comprise the adult and neonatal forms of MAP2 and tau will be purified and individual polypeptides characterized by two-dimensional peptide mapping before and after phosphorylation by either purified cAMP-dependent or calcium-calmodulin-dependent protein kinase. Light and electron microscopic immunocytochemistry will be performed with an existing panel of monoclonal antibodies to identify the cellular and subcellular distribution of these two proteins in adult and developing brain. Monoclonal antibodies will be produced which exclusively recognize either individual electrophoretic species of MAP2 or tau or differentially phosphorylated forms of these two proteins. These monoclonal antibodies will be used to study the cellular and subcellular distribution of specific isoforms of MAP2 and tau.
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