We are proposing to investigate an ocular lens cytoskeletal structure known as the beaded filament. This structure, and the two proteins thus far associated with it, have not been demonstrated in either the lens epithelium, or in other tissues. Thus the beaded filament appears to be a structure unique to the differentiating lens fiber cell. For this reason we feel that the beaded filament must be of seminal importance to normal lens biology. We are proposing to use subcellular fractionation to purify the beaded filament, and implicate, by co-purification, any proteins which might be associated with this structure. Monoclonal antibodies to candidate proteins will be used to confirm the presence of specific proteins in the beaded filament. We propose to establish conditions in which the beaded filament can be reconstituted, initially from disrupted whole filaments, and subsequently from purified individual constituent proteins. Using either the reconstitution system developed herein, or a cell-free translation system programmed with lens mRNA, filament assembly will be specifically disrupted, using F(ab) fragments of monoclonal antibodies, cyanogen bromide fragments of beaded filament proteins, synthetic peptides derived from the primary sequence, or oligonucleotides synthesized from primary sequence information. In so doing we hope to pinpoint protein domains involved in beaded filament protein-protein interactions. We intend to establish the primary amino acid sequence for the proteins which comprise the beaded filament, by isolating the cDNA for these proteins from existing cDNA libraries. We plan to utilize this sequence information to relate the proteins to existing gene families, and for subsequent investigation of specific binding domains within beaded filament proteins. Subsequent inquiry will be made into the possibility of utilizing a differentiating lens explant for further in vitro studies of beaded filament transcription and translation. In vitro synthesis of beaded filaments in a cell-free translation system will be employed to study filament protein synthesis and assembly. Organ culture of whole lenses, as well as in vitro synthesis will be used to study the phosphorylation of the beaded filament proteins, the regulation of this phosphorylation, and the effects of phosphorylation on filament assembly and disassembly. We believe that such basic knowledge of the system which generates and maintains cellular shape and tissue level organization in the lens is fundamental to understanding how the lens achieves and maintains clarity, and permits us to study the possible role of the cellular cytoskeleton in the etiology of cataract.
| Chernyatina, Anastasia A; Hess, John F; Guzenko, Dmytro et al. (2016) How to Study Intermediate Filaments in Atomic Detail. Methods Enzymol 568:3-33 |
| 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 |
| 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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