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.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
Project #
Application #
Study Section
Visual Sciences A Study Section (VISA)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Northwestern University at Chicago
Schools of Dentistry
United States
Zip Code
Rafferty, N S; Rafferty, K A; Zigman, S (1997) Comparative response to UV irradiation of cytoskeletal elements in rabbit and skate lens epithelial cells. Curr Eye Res 16:310-9
Zigman, S; Rafferty, N S (1994) Catalase activity in dogfish (Mustelus canis) ocular tissues. Biol Bull 187:247-8
Zigman, S; Rafferty, N S (1994) Effects of near UV radiation and antioxidants on the response of dogfish (Mustelus canis) lens to elevated H2O2. Comp Biochem Physiol Physiol 109:463-7
Rafferty, N S; Rafferty, K A; Ito, E (1994) Agonist-induced rise in intracellular calcium of lens epithelial cells: effects on the actin cytoskeleton. Exp Eye Res 59:191-201
Rafferty, N S; Zigman, S; McDaniel, T et al. (1993) Near-UV radiation disrupts filamentous actin in lens epithelial cells. Cell Motil Cytoskeleton 26:40-8
Zigman, S; Rafferty, N S; Scholz, D L et al. (1992) The effects of near-UV radiation on elasmobranch lens cytoskeletal actin. Exp Eye Res 55:193-201
Rafferty, N S; Rafferty, K A (1992) Lens cytoskeleton and after-cataract. Acta Ophthalmol Suppl :34-45
Rafferty, N S; Scholz, D L (1991) Development of actin polygonal arrays in rabbit lens epithelial cells. Curr Eye Res 10:637-43
Rafferty, N S; Scholz, D L; Goldberg, M et al. (1990) Immunocytochemical evidence for an actin-myosin system in lens epithelial cells. Exp Eye Res 51:591-600
Liou, W (1990) Whole-mount preparations of mouse lens epithelium for the fluorescent cytological study of actin. J Microsc 157:239-45

Showing the most recent 10 out of 16 publications