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Confocal Microscopy Confirmed that in Phosphatidylcholine Giant Unilamellar Vesicles with very High Cholesterol Content Pure Cholesterol Bilayer Domains Form

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Abstract

The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. This high Chol content forms pure Chol bilayer domains (CBDs) and Chol crystals in model membranes and membranes formed from the total lipid extracts from human lenses. CBDs have been detected using electron paramagnetic resonance (EPR) spin-labeling approaches. Here, we confirm the presence of CBDs in giant unilamellar vesicles prepared using the electroformation method from Chol/1-palmitoyl-2-oleoylphosphocholine and Chol/distearoylphosphatidylcholine mixtures. Confocal microscopy experiments using phospholipid (PL) analog (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid) and cholesterol analog fluorescent probes (23-(dipyrrometheneboron difluoride)-24-norcholesterol) were performed, allowing us to make three major conclusions: (1) In all membranes with a Chol/PL mixing ratio (expressed as a molar ratio) >2, pure CBDs were formed within the bulk PL bilayer saturated with Chol. (2) CBDs were present as the pure Chol bilayer and not as separate patches of Chol monolayers in each leaflet of the PL bilayer. (3) CBDs, presented as single large domains, were always located at the top of giant unilamellar vesicles, independent of the change in sample orientation (right-side-up/upside-down). Results obtained with confocal microscopy and fluorescent Chol and PL analogs, combined with those obtained using EPR and spin-labeled Chol and PL analogs, contribute to the understanding of the organization of lipids in the fiber cell plasma membranes of the human eye lens.

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Acknowledgements

This work was supported by grants EY015526 and EY001931 from the National Institutes of Health, USA.

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  1. Laxman Mainali

    Present address: Department of Physics, Boise State University, 1910 University Drive, Boise, Idaho, 83725, USA

Authors and Affiliations

  1. Department of Medical Physics and Biophysics, School of Medicine, University of Split, Split, Croatia

    Marija Raguz

  2. Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA

    Marija Raguz, Mariusz Zareba, Laxman Mainali & Witold K. Subczynski

  3. Department of Pathology, CRI Imaging Core, Translational and Biomedical Research Center, Medical College of Wisconsin, Wauwatosa, WI, USA

    Suresh N. Kumar

  4. Department of Ophthalmology, Medical College of Wisconsin Eye Institute, Milwaukee, WI, USA

    Mariusz Zareba

  5. Department of Physics, Faculty of Science, University of Split, Split, Croatia

    Nada Ilic

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Raguz, M., Kumar, S.N., Zareba, M. et al. Confocal Microscopy Confirmed that in Phosphatidylcholine Giant Unilamellar Vesicles with very High Cholesterol Content Pure Cholesterol Bilayer Domains Form. Cell Biochem Biophys 77, 309–317 (2019). https://doi.org/10.1007/s12013-019-00889-y

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