Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach

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Abstract

Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function. Cholesterol is the most representative sterol present in higher eukaryotes. It is often found distributed non-randomly in domains or pools in biological and model membranes. Cholesterol-rich functional microdomains (lipid rafts) are often implicated in cell signaling and membrane traffic. Interestingly, lipid rafts have also recently been isolated from organisms such as yeast and Drosophila, which have ergosterol as their major sterol component. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is not very clear. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing the environment-sensitive fluorescent membrane probe DPH. Our results from steady state and time-resolved fluorescence measurements show, for the first time, differential effects of ergosterol and cholesterol toward membrane organization. These novel results are relevant in the context of lipid rafts in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.

Section snippets

Materials and methods

Materials. DPPC and POPC were obtained from Avanti Polar Lipids (Alabaster, AL, USA). DMPC, cholesterol, ergosterol, and DPH were purchased from Sigma Chemical (St. Louis, MO, USA). All lipid stock solutions were made in methanol except ergosterol which was dissolved in ethanol. Phospholipids were checked for purity by thin layer chromatography on pre-coated silica gel plates (Sigma) in chloroform/methanol/water (65:35:5, v/v/v) and were found to give only one spot in all cases with a

Results

The change in fluorescence polarization of DPH with increasing sterol concentration is shown in Fig. 2. Fluorescence polarization is correlated to the rotational diffusion rate [25] of membrane embedded probes which is sensitive to the packing of fatty acyl chains and sterols. Fig. 2A shows that with increase in cholesterol concentration, the DPH polarization in fluid POPC membranes shows a continuous increase up to the highest concentration of cholesterol used. Thus, there is large (123%)

Discussion

Cholesterol is the major sterol molecule ubiquitously present in mammalian cells. It is hypothesized that cholesterol is selected in the long natural evolution process for its ability to optimize certain physical requirements of cell membranes (such as packing) in relation to functions performed by the cell [1], [38], [39]. Cholesterol appears to be the appropriate molecule to maintain a delicate balance between membrane rigidity to allow for large cell volumes (as found in higher eukaryotes)

Acknowledgements

This work was supported by the Council of Scientific and Industrial Research, Government of India. We gratefully acknowledge Satinder Rawat for preliminary experiments. We thank Y.S.S.V. Prasad and G.G. Kingi for technical help, and members of our laboratory for critically reading the manuscript. A.A. acknowledges the Indian Council of Medical Research for the award of a Project Assistantship. H.R. thanks the Council of Scientific and Industrial Research for the award of a Senior Research

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    Abbreviations: DMPC, dimyristoyl-sn-glycero-3-phosphocholine; DPH, 1,6-diphenyl-1,3,5-hexatriene; DPPC, dipalmitoyl-sn-glycero-3-phosphocholine; MLV, multilamellar vesicle; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

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