Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes

Abstract

The purpose of this study was to generate the equivalent of a cholesterol/temperature phase map for a biological membrane using fluorescence spectroscopy. The pseudo-phase map was created using human erythrocytes treated with various concentrations of methyl-b-cyclodextrin to remove defined amounts of cholesterol and a trio of fluorescent probes that assess different membrane properties (laurdan, diphenylhexatriene, and merocyanine 540). Parallel experiments with two-photon microscopy suggested that changes in cellular cholesterol content affected the entire membrane rather than being localized to specific macroscopic domains. The various regions of the composite erythrocyte pseudo-phase map were interpreted using analogous data acquired from multilamellar vesicles that served as simplified models of cholesterol-dependent phases. The vesicles consisted of various concentrations of cholesterol (0 to 50 mol%) with either palmitoyl sphingomyelin, 1:1 dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine, or phospholipid mixtures intended to simulate either the inner or outer leaflet of erythrocyte membranes. Four distinguishable regions were observed in sphingomyelin phase maps corresponding to the traditional solid-ordered and liquiddisordered phases and two types of liquid-ordered behavior. Physical properties were less diverse in the mixed phospholipid vesicles, as expected, based on previous studies. Erythrocytes displayed five regions of different combinations of membrane properties along the phase map. Some of the observations identified similarities between the cells and liquidordered behavior observed in the various types of liposomes as well as some interesting

Structural organization of plasma membrane lipids isolated from cells cultured as a monolayer and in tissue-like conditions

“…The increased content of cholesterol and sphingomyelin in the 3D plasma membrane lipid bilayer as well the higher PE (low hydrated polar headgroup) and PS (charged polar headgroup) levels are factors which determine an increased GP value. The changes in GP are clearly correlated with the amount of cholesterol in the range of 0.2-0.4 mol% [32,33] as it is consistent with our data. In addition, the presence of anionic phospholipids leads also to blue-shifted specters and increased GP values as a function of the charged lipids concentration [34].…”

Relationship between membrane permeability and specificity of human secretory phospholipase A2 isoforms during cell death

“…Dipalmitoylphosphatidylglycerol (DPPG) was obtained from Avanti Polar Lipids (Birmingham, AL). Multilamellar liposomes were prepared by hydration and vortex agitation of dried lipid samples at 50 °C as described [31]. …”

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

“…Depletion of cholesterol from living fibroblasts or CHO cells labeled by fluorescent SM analogs induces the formation of submicrometric domains or increases their size, indicating a restricting role of cholesterol for domain formation/maintenance in these cells [30, 173]. In contrast, slight cholesterol depletion of the RBC PM decreases the abundance of PC- and SM- but not GSLs-enriched submicrometric domains [26, 27] as well as lipid packing, as revealed by Laurdan [185]. Moreover, cholesterol influences the shape of submicrometric domains.…”