This is a collaborative proposal between the laboratories of David R. Colman at Mount Sinai, NY, and of Graciela L. Boccaccio at the Institute Leloir (formerly Institute for Biochemical Research Campomar Foundation), Buenos Aires, Argentina. This proposal extend our ongoing work on the Functional Organization of the Myelin Axoglial Junction (grant # 1R01 NS40560; 07/01/00 - 06/30/05) to the study of intracellular elements of the glial cell cytoplasm adjacent to the junction. Specifically, we are aimed to elucidate the cell mechanisms mediating the localized synthesis of proteins in the myelinating cells of the central nervous system, that has being for long time of major interest to both, the applicant's and the foreign collaborator's research group. We will investigate the participation of the double stranded RNA binding protein Staufen in the localization and translation of mRNAs in oligodendrocytes. This is likely relevant to two related processes: 1. synthesis of components required for membrane maintenance and remodeling, and 2, signal transduction at the oligodendrocyte side of the axoglial junction. We will 1) use mass spectrometry to unambiguously identify novel protein components of the mRNA localization apparatus and 2) begin to asses the role of Staufen and novel components in mRNA localization. An important aspect of this proposal is its complementary nature combining the scientific interests and the methodological strength of two laboratories. Dr. Boccaccio's group have developed primary culture of oligodendrocytes and biochemical analysis of brain subcellular components, as well as a functional assay for mRNA localization in cultured cells. A number of methods that are strictly required for the present project have been established in our laboratory in Mount Sinai, namely, protein analysis via mass spectrometry and analysis of protein-protein interactions by yeast two-hybrid and """"""""pull-down"""""""" experiments. Thus, this project will allow the training of the foreign investigator and Ph.D. students on state-of-the-art techniques that are currently not established at their home institute, thus representing a valuable contribution
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