I propose to characterize the morphology and map the distribution of cell fusion in normal adult frog and rat lenses. The gross surface morphology, size and length of fusion between lens fibers will be examined by scanning electron microscope (SEM). In addition, the distribution of fiber fusions as a function of radial location and as a function of location along fiber length will be analyzed by SEM. Correlative SEM and light microscopy will be employed to estimate the total number of fiber fusions per lens. Freeze fracture replicas of fiber fusions will be prepared to reveal the ultrastructure of membrane between fused cells in situ. Freeze fracture will also be used to estimate the distribution of gap junctions throughout lenses. These estimates will be compared with the distribution of fiber fusions to determine if there is any relationship between these two types of intercellular contact. Lenses will also be prepared for examination by light voltage electron microscopy (HVEM). Lenses for HVEM selectively stained to enhance membrane should reveal if lateral and long membrane of fibers are equally involved in cell fusion. Lenses for HVEM selectively stained to enhance the extracellular space should demonstrate any gap junctional involvement in fiber fusion and reveal a view of fused fibers that can be correlated with the SEM observations. Lenses for HVEM will also be examined to study the lens cytoskeleton between fused fibers. At least two types of cataract (traumatic, trans-corneal needle injury, Fagerholm and Philipson, 1979, and congenital cataract webbed peromyscus deer mouse lens, Feeney et al. 1980) are characterized by gross examples of fiber fusion. The sites of opacity in these lenses correspond to areas in the lens that feature several factors that have been shown to affect cell fusion in other systems. The results of this study will be a more comprehensive knowledge of normal lens morphology. This will enable future studies to assess more accurately the changes in morphology that occur during cataractogenesis.
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