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PV Rao, RL Maddala, PF Deng, MJ Costello, E Wawrousek, JS Zigler; Cytoskeletal and Morphological Changes in Rho GTPase Inactivated Transgenic Mouse Lens . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1923.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Using a transgenic mouse expressing the Rho GTPase inhibitor (C3-exoenzyme) specifically in lens tissue, we have recently demonstrated the importance of Rho GTPase in lens growth and function. The purpose of this study is to utilize the C3 transgenic mouse to characterize cytoskeletal and morphological changes caused by inhibition of the Rho GTPase signaling pathway. Methods:Eyes obtained from 1 and 7 day old C3 transgenic and normal littermate mice were fixed either for cryosectioning or transmission electron microscopy (TEM) analysis. Whole eye cryosections were stained for F-actin (rhodamine phalloidin) or immunostanied for focal adhesion proteins (vinculin or paxillin) and adherens junctional proteins (ß-catenin or pan cadherin) with FITC, TRITC or peroxidase conjugated secondary antibodies and confocal (Zeiss) images were recorded. Thin sections stained for morphological changes were photographed using a Philips TEM. Results:In wild type mouse lenses at days 1 and 7, F-actin (phalloidin) was distributed throughout including epithelium, equatorial fibers, cortical and central fibers with intense staining at the interface of anterior fibers and lens epithelium, and posterior fiber ends and the capsule. Vinculin and paxillin stained intensely at the equatorial fibers, epithelium and posterior fiber ends. The epithelium and equatorial and cortical fibers stained strongly for ß-catenin and cadherins. In contrast to normal lenses, fiber cells from transgenic lenses were grossly distorted and disorganized, exhibiting abnormal cell shape, thickness and membrane architecture. Staining for F-actin, vinculin, paxillin, ß-catenin and cadherins was reduced dramatically. TEM analysis also confirmed that as compared to littermate controls, elongating fibers in transgenic lens were severely distorted in size and shape with large extracellular spaces between the fibers. Although the lens epithelium appeared normal, lens capsule thickness was markedly increased in the transgenic mice. Conclusion:The immunostaining of cytoskeletal proteins together with morphological changes observed in C3-transgenic lenses demonstrate that Rho GTPase inactivation impairs cytoskeletal organization and distorts fiber cell shape, organization and migration leading to lens opacity.
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