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.
| Cheng, Catherine; Nowak, Roberta B; Biswas, Sondip K et al. (2016) Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells. Invest Ophthalmol Vis Sci 57:4084-99 |
| Chernyatina, Anastasia A; Hess, John F; Guzenko, Dmytro et al. (2016) How to Study Intermediate Filaments in Atomic Detail. Methods Enzymol 568:3-33 |
| Sun, Ning; Shibata, Brad; Hess, John F et al. (2015) An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies. Mol Vis 21:428-42 |
| Sindhu Kumari, S; Gupta, Neha; Shiels, Alan et al. (2015) Role of Aquaporin 0 in lens biomechanics. Biochem Biophys Res Commun 462:339-45 |
| Gerhart, Jacquelyn; Greenbaum, Marvin; Scheinfeld, Victoria et al. (2014) Myo/Nog cells: targets for preventing the accumulation of skeletal muscle-like cells in the human lens. PLoS One 9:e95262 |
| Stewart, Daniel N; Lango, Jozsef; Nambiar, Krishnan P et al. (2013) Carbon turnover in the water-soluble protein of the adult human lens. Mol Vis 19:463-75 |
| Fan, Jianguo; Dong, Lijin; Mishra, Sanghamitra et al. (2012) A role for ?S-crystallin in the organization of actin and fiber cell maturation in the mouse lens. FEBS J 279:2892-904 |
| Castorino, John J; Gallagher-Colombo, Shannon M; Levin, Alex V et al. (2011) Juvenile cataract-associated mutation of solute carrier SLC16A12 impairs trafficking of the protein to the plasma membrane. Invest Ophthalmol Vis Sci 52:6774-84 |
| 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 |
| Shi, Yanrong; De Maria, Alicia B; Wang, Huan et al. (2011) Further analysis of the lens phenotype in Lim2-deficient mice. Invest Ophthalmol Vis Sci 52:7332-9 |
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