The proposed research will test the hypothesis that the arrangement and composition of filaments of the cytoskeleton of lens epithelial and fiber cells determine lens shape and accommodaive ability, and conversely, that disarrangement of the cytoskeleton is correlated with presbyopia and/or aging cataract. Since rebound to the more spherical shape from the flattened state in a lens with significant accommodative amplitude is not understood and cannot be explained by capsular elasticity alone, the role of the cytoskeleton in this process is proposed to explain inherent lens elasticity. The approaches proposed to test the hypothesis include comparative morphological and bio-chmeical studies of lenses from accommodating (monkey) and non-accommodating (rabbit, mouse) species, using young and old members, and whose lens shape has been modified. The methodologies to be used to test the hypothesis will focus on the recently discovered polygonal arrays of microfilaments associated with the cytoplasmic surface of the apical epithelial plasma membrane. Since similar arrays have been shown in a variety of cultured cells to contain contractile proteins, identification and localization of these proteins in whole lens tissue will be pursued. 1) Antibodies against actin, myosin, Alpha-actinin and filamin will be raised in rabbits, purified by column chromatography and used to identify the proteins by immunofluorescence light microscopy of lens epithelial whole mounts and sections of embedded lenses, and by immunogold electron microscopy of thin sections. Antivimentin monoclonal antibody will be used similarly to localize this intermediate filament protein in epithelial cells; 2) These same antibodies will be used in Western immuno-blotting techniques in conjunction with SDS-PAGE of lens extracts, to verify the identification of the contractile proteins; 3) Cycloplegic and cholinomimetic drugs and a mechanical device will be used on rabbit and monkey eyes to change lens shape, and these will be compared with regard to numbers of polygonal arrays, intervertex distance, and distribution of contractile proteins. In addition to immunofluorescence and immunogold techniques, fluorescence microscopy of rhodamine-phalloidin labeled epithelial whole mounts will be used; 4) Morphologic studies with high-voltage electron microscopy will be used to detemrine the depth and connections of the polygonal arrays to other cellular organelles; 5) Morphologic study of the vertex of the polygonal array will be made using freeze-fracture and high magnification TEM in order to determine its structure.
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