Long-term objectives of the proposed research are to test the hypothesis that the lens cytoskeleton and its associated proteins are involved in maintaining lens shape, in lens accommodation and development of presbyopia, and in formation of certain kinds of cataract when the cytoskeleton is disrupted.
One specific aim i s to assess the role of cytosolic free calcium on homeostasis of the lens cytoskeleton.
A second aim i s to determine whether changes in the ratio of soluble (C) actin to filamentous (F) actin in lens cells occurs during aging. Changes in calcium levels in lenses of different ages will also be monitored, since certain actin-binding proteins that polymerize actin and attach actin filaments to plasma membranes are affected by intracellular calcium. if lens actin shifts heavily to the polymerized form it may resist accommodative lens shape changes, resulting in presbyopia.
A third aim i s to study the effect of ultraviolet light on the lens cytoskeleton and to determine the target that causes disruption of the cytoskeleton. The effect of UV light on intracellular calcium levels in lens epithelial cells will be studied, to determine whether UV disruption of the cytoskeleton operates indirectly through an effect on calcium levels. Methods to be used are DNAase I inhibition assay to find the ratios of G/F actin; fluorometric measurement of calcium concentrations in epithelia of lenses treated with calcium-channel blockers, calcium-ATPase inhibitors, inositol trisphosphate, lowered extracellular calcium, and mechanical stretching. Measurements are made using fluorescent calcium indicators (Fluo-3 and Fura-2) loaded into lens epithelial cells in vitro. Other methods used to identify proteins and assess changes resulting from aging, changing intracellular calcium concentrations, and UV irradiation damage are SDS-PAGE and immunoblotting, immunofluorescence microscopy, and immunogold transmission and scanning electron microscopy. Antibodies against spectrin, ankyrin, vinculin, tubulin and vimentin will be used in these studies. Animals: Rabbits.
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