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Confocal Microscopy Confirmed that in Phosphatidylcholine Giant Unilamellar Vesicles with very High Cholesterol Content Pure Cholesterol Bilayer Domains Form. Cell Biochem Biophys 2019 Dec;77(4):309-317

Date

10/19/2019

Pubmed ID

31625023

Pubmed Central ID

PMC6858498

DOI

10.1007/s12013-019-00889-y

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.

Author List

Raguz M, Kumar SN, Zareba M, Ilic N, Mainali L, Subczynski WK

Authors

Suresh Kumar PhD Associate Professor in the Pathology department at Medical College of Wisconsin
Witold K. Subczynski PhD Professor in the Biophysics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Cholesterol
Electron Spin Resonance Spectroscopy
Fluorescent Dyes
Humans
Lens, Crystalline
Lipid Bilayers
Microscopy, Confocal
Phosphatidylcholines
Unilamellar Liposomes
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a