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W-K Lo, CJ Zhou, M Scanlon; Depletion of Cholesterol Abolishes Fiber Cell Interlocking Membrane Domains in the Rat Lens . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3998.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cholesterol is enriched in lipid microdomains of plasma membranes in various cell types. It has been shown that membrane cholesterol plays an important role in the formation and maintenance of membrane curvatures and protrusions needed for specific cell functions. Since lens fiber cells are unique in possessing both high membrane cholesterol and extensive interlocking membrane domains (protrusions) in all species, we investigated the relation between cholesterol-rich domains and protrusions by examining the cell surface changes associated with cholesterol depletion in organ-cultured lens. Methods: Freshly isolated rat lenses (3-5 weeks old) were pre-conditioned in an incubator in medium 199 plus antibiotics for at least 4 hours at 37 C. Undamaged clear lenses were then incubated in control medium and medium enriched with 10 mM methyl-ß-cyclodextrin (MBCD) for 4, 8, 16 and 24 hrs. Lenses were processed for scanning EM and fluorescence labeling of cholesterol on frozen sections using 0.05 mg/ml filipin solution and examined with a microscopic imaging system equipped with a UV light source. Results: All treated lenses exhibited cortical opacity after a 4-hr incubation in 10 mM MBCD whereas the control lenses remained transparent. The density of cortical opacity was progressively increased as a function of incubation time. SEM studies from lens halves with good fiber orientation showed that protrusions of superficial fiber cells, approximately 30 fiber cells deep from the capsule surface, disappeared completely after a 4-hr MBCD treatment. The protrusion-free zone was extended deeper with increased incubation time (e.g., ∼120 cells deep after 24-hr treatment). The gradual disappearance of protrusions was noticeable in the area between protrusion-free and protrusion-rich zones, suggesting that the depletion of cholesterol and protrusion domains was caused by exposure to MBCD. This was further supported by the weak or absent cholesterol labeling by filipin in the superficial protrusion-free zone, as compared with the strong punctate labeling pattern along cell membranes of the deeper protrusion-rich zone. Conclusion: Depletion of membrane cholesterol by methyl-ß-cyclodextrin is associated with the disappearance of interlocking membrane domains in cortical fiber cells. This result suggests that cholesterol may play a role in the formation and maintenance of interlocking domains of lens fiber cells.
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