engleski

Colesterol Bilayer Domain in Phospholipid Bilayer Membranes Can be Detected by Confocal Microscope

The most unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content ; cholesterol/phospholipid molar ratio can be as high as 4 in human lens nucleus. Cholesterol saturates the bulk phospholipid bilayer and induces formation of immiscible cholesterol bilayer domains (CBDs) within the membrane. The presence of a CBD plays a crucial role ensuring that the surrounding phospholipid bilayer is saturated with cholesterol which keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly with age. Our previous experiments were based on electron paramagnetic resonance spin-labeling methods. They allowed detection of CBDs in model and lens lipid membranes at high cholesterol content1, 2, 3. In current experiments the giant unilamellar vesicles made of cholesterol / distearoylphosphatidylcholine (Chol / DSPC) mixtures, with mixing ratio from 0.5 to 5 and labeled with phospholipid analog (1, 1'- Dioctadecyl-3, 3, 3', 3'-Tetramethylindocarbocyanine- 5, 5'-Disulfonic Acid) and cholesterol analog (23- (dipyrrometheneboron difluoride)-24- norcholesterol) fluorescent probes, were prepared using the electroformation method4. Confocal microscopy experiment allowed us to confirm that membranes with higher Chol / DSPC molar ratio contain two distinct lipid environments, the bulk phospholipid-cholesterol domain (PCD) and the CBD. The amount of CBD was greater in membranes with higher Chol / DSPC ratio. According to these results, CBD is always located on the top of these giant vesicles possibly because CBD has smaller mass per unit area of the vesicle compared with PCD domain. Additionally, it can be concluded that vesicles with higher Chol/DSPC molar ratio are more rigid and show less shape fluctuation in water environment.

eye lens; fiber cell; plasma membrane; cholesterol; giant umilamellar vesicles

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