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W.–K. Lo, W. Peng, C. Zhou, L. Brako, S. Biswas; A Unique Distribution of Lipid Raft Proteins Is Associated With Interlocking Membrane Domains During Lens Fiber Cell Differentiation and Maturation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1865.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The structure and function of lipid rafts in lens fiber cells are largely unknown. We have hypothesized that interlocking membrane domains (IMD) are the major sites of lipid rafts in fiber cells, based on our preliminary findings that depletion of membrane cholesterol by methyl–beta–cyclodextrin abolished IMD in rat lens in organ culture, and the cholesterol labeling by filipin exhibited a dotted pattern along fiber cell membranes. To further test this hypothesis, we examined the presence and specific distribution of a number of key lipid raft proteins in lens fiber cells. Methods: Immunofluorescence labeling of various lipid raft antibodies was conducted on paraffin or frozen sections of rat and chicken embryonic lenses. Immunogold EM labeling of cav–1 was performed in chicken lens. Western blotting was carried out in crude membrane fractions of lens fibers of various species. Results: Immunofluorescence analyses showed that dotted labeling patterns of flotillin 1 and 2, annexin II, Thy–1 (GPI–anchored protein), c–Src kinase and plasma membrane Ca+2–ATPase were regularly found associated with the corners of hexagonal fiber cells in the rat lens where IMD are generally located. Of particular interest is the caveolin–1 localization in chicken embryonic lenses. Cav–1 was found initially labeled along the entire cell membranes in the superficial young fiber cells, and its labeling was gradually shifted toward the corners of the cells and eventually became concentrated only in the finger–like IMD at the corners of deeper cortical fiber cells. SEM study revealed that numerous finger–like IMD were readily observed at the corners of inner cortical fibers in chicken lenses. These results suggest that cav–1 is synthesized in differentiating young fiber cells and is gradually recruited to IMD during fiber cell maturation. Immunogold EM confirmed that cav–1 was localized on IMD of chicken lens fiber cells. Western blot detected the presence of all tested lipid raft proteins in crude membrane fractions of fiber cells from various species, except that caveolins and flotillins showed species variations. Conclusions: A preferential dotted pattern of antibody labeling at the corners of fiber cells for all lipid raft proteins studied here suggests that these proteins are associated with lens–specific interlocking membrane domains. A unique distribution of cav–1 in chicken embryonic lens provides us the best model for studying the structural and functional roles of caveolin in relation to the development of cholesterol–rich lipid raft interlocking domains in the lens.
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