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Cell Biochemistry and Biophysics

, Volume 77, Issue 4, pp 309–317 | Cite as

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

  • Marija RaguzEmail author
  • Suresh N. Kumar
  • Mariusz Zareba
  • Nada Ilic
  • Laxman Mainali
  • Witold K. SubczynskiEmail author
Original Paper
  • 76 Downloads

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.

Keywords

Confocal microscopy Cholesterol Cholesterol bilayer domain Membrane Giant unilamellar vesicles 

Notes

Acknowledgements

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

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  • Marija Raguz
    • 1
    • 2
    Email author
  • Suresh N. Kumar
    • 3
  • Mariusz Zareba
    • 2
    • 4
  • Nada Ilic
    • 5
  • Laxman Mainali
    • 2
    • 6
  • Witold K. Subczynski
    • 2
    Email author
  1. 1.Department of Medical Physics and Biophysics, School of MedicineUniversity of SplitSplitCroatia
  2. 2.Department of BiophysicsMedical College of WisconsinMilwaukeeUSA
  3. 3.Department of Pathology, CRI Imaging Core, Translational and Biomedical Research CenterMedical College of WisconsinWauwatosaUSA
  4. 4.Department of OphthalmologyMedical College of Wisconsin Eye InstituteMilwaukeeUSA
  5. 5.Department of Physics, Faculty of ScienceUniversity of SplitSplitCroatia
  6. 6.Department of PhysicsBoise State UniversityBoiseUSA

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