Support is requested for continued studies of a lens fiber cell cytoskeletal element, the Beaded Filament, and two proteins, """"""""CP49"""""""" and """"""""CP115"""""""", which comprise it. Both the Beaded Filament, and the two proteins which comprise it, are unique to the lens fiber cell, and, therefore presumed to be of critical importance to normal lens biology. The potential importance of the beaded filament to the lens and eye is underscored by the recent revelation that the Elo mouse line, a naturally- occurring, autosomal dominant strain with inherited microphthalmia, has been shown to lack a single protein: CP115. We propose to study the Beaded Filament at three levels: I) GENE STRUCTURE OF BEADED FILAMENT PROTEINS: We will define the structure of the genes for the lens-specific CP49 and CP115, establishing intron size, number, and location. We propose to define the location/sequence of upstream regions which restrict expression to the lens fiber cell. Such studies will a) further clarify the relationship between the Beaded Filament and other cytoskeletal elements, b) shed light on promoters which target proteins to the lens fiber cell, and c) permit transfection, transgenic, and ectopic expression studies on these two proteins. II) STRUCTURE OF THE BEADED FILAMENT, AND BEADED FILAMENT PROTEINS: We will isolate and sequence CP49 and CP115 cDNAs from divergent sources. These will be used to conduct a comparison of these cDNAs, the translated protein sequences, and the predicted secondary structure. This comparison will identify highly conserved regions of the beaded filament proteins, and therefore permit: a) construction of a """"""""consensus"""""""" model for the beaded filament proteins, b) provide a rational basis for studies, and the interpretation of studies which empirically test the model, studies which include: proteolysis, immunolabelling, and cross-linking of filaments, and filament proteins, production of normal recombinant proteins, and of deletion/site directed mutant proteins for structural and assembly studies. III) CELL/TISSUE BIOLOGY OF THE BEADED FILAMENT: To explore the role of the beaded filament in the lens cell, several questions will be asked: 1) Does the BF have special relationship or attachment to any cell structure(s) or membrane domains? 2) What is the relationship between the beaded filament and the intermediate filament? 3) What impact does the age-related degradation of BF proteins have on beaded filament structural integrity, and what is the mechanism by which this degradation occurs?
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| Fudge, Douglas S; McCuaig, John V; Van Stralen, Shannon et al. (2011) Intermediate filaments regulate tissue size and stiffness in the murine lens. Invest Ophthalmol Vis Sci 52:3860-7 |
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