MOLECULAR ANALYSIS OF OCULAR LENS BEADED FILAMENTS

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DESCRIPTION: The beaded filament is a structurally unique cytoskeletal element, composed of two proteins expressed only in the fiber cell of the ocular lens (CP49 and CP115). As the expression of Beaded Filament proteins is turned on, the expression of other cytoskeletal proteins is turned off. This ultimately leaves the Beaded Filament as the dominant, and perhaps sole cytoskeletal structure in the mature fiber cell. In the previous funding period the PI established much about the sequence and structure of the Beaded Filament proteins. This information provides a rationale basis for asking questions designed to: establish the function of the Beaded Filament in the lens; determine the importance of the Beaded Filament to lens biology; determine the molecular mechanisms of Beaded Filament protein assembly; establish how, as extreme members of the Intermediate Filament family, the Beaded Filament has utilized unique modifications to primary sequence and secondary structure to adapt to lens-specific functions. The PI proposes three major avenues of investigation: I. What is/are the role(s) of the Beaded Filament in the biology of the lens fiber cell? He will employ ultrastructural, biochemical, and genetic approaches, including germline "knock out/knock in" strategies. II. What are the molecular interactions between the two Beaded Filament proteins? How do they assemble, and what is the impact of the unique changes seen in their primary sequence and secondary structure? He will use expressed proteins, Yeast Two Hybrid analysis, assembly studies, cross-linking, analytical chromatography, and mutational analysis. III. What mechanisms are used to regulate the levels of Beaded Filament proteins in the cytoplasm of the lens fiber cell? How is the stoichiometry of these proteins kept in balance, and what is the role of Beaded Filament protein phosphorylation? He will use germline knockouts, analysis of protein and mRNA levels, transfection, and assembly studies.