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Laxman Mainali, William J O'Brien, Witold Karol Subczynski; Detection of cholesterol bilayer domains in intact membranes of eye lens fiber cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3033.
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© ARVO (1962-2015); The Authors (2016-present)
In the plasma membranes of the eye lens fiber cells, which are loaded with cholesterol and dense with integral membrane proteins, four purported lipid domains are expected. Three of these domains, namely the bulk, boundary, and trapped lipids, have been detected. The cholesterol bilayer domain (CBD) has been discriminated in the lens lipid membranes (LLMs) prepared from the total lipid extracts from fiber cell plasma membranes, but it has not yet been detected in intact fiber cell plasma membranes. It is hypothesized that, when CBDs are detected in LLMs, they also exist in intact lens membranes. To prove or disprove that hypothesis, a method allowing detection of CBDs in intact membranes was developed.
A saturation-recovery (SR) electron paramagnetic resonance (EPR) spin-labeling method was developed that allows identification of CBDs in intact membranes. It is a modification of the previously developed SR EPR method (named discrimination by oxygen transport) and is based on the SR signal measurements of the cholesterol-analog spin label located in the lipid bilayer portion of intact membranes as a function of the partial pressure of the molecular oxygen with which the samples are equilibrated.
The capabilities and limitations of the developed method were illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. CBDs were detected only in porcine nuclear intact membranes, but not in porcine cortical intact membranes. CBDs were also detected only in porcine nuclear LLMs, but not in porcine cortical LLMs. CBDs were detected in both intact nuclear and cortical fiber cell plasma membranes isolated from the lenses of 40-, 46-, and 53-year-old human donors with cholesterol content high enough to induce formation of CBDs in LLMs.
We conclude the following: (1) CBDs, when present, can be detected in intact fiber cell plasma membranes using the developed SR EPR spin-labeling method. (2) The obtained results support the hypothesis that, when CBDs are detected in LLMs, they also exist in intact lens membranes. (3) The results obtained for intact membranes, in combination with those obtained for LLMs, contribute to the understanding of the organization of lipids in the fiber cell membranes of the human eye lens (loaded with cholesterol and dense with integral membrane proteins).
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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