Sterol Properties Required for Microdomain Formation: From Model Systems to Living Yeast and Mammalian Cells

Abstract

This work presents new insights into the evolution of sterol-enriched domains in the plasma membrane (PM) of eukaryotes. The recent finding of sphingolipid-enriched gel domains in the PM of Saccharomyces cerevisiae(1) demands novel strategies to study sterol-enriched liquid ordered domains in this organism. Therefore, we employed di-4-ANEPPS fluorescence, here shown to be sensitive to S. cerevisiae sterols and Fluorescence Lifetime Imaging Microscopy (FLIM) for the first time in S. cerevisiae PM. In vivo studies were performed with S. cerevisiae (wild type and erg6Δ), and animal (CHO) cells, modeled by liposomes containing ergosterol, zymosterol, or cholesterol, respectively. Different fluorescent probes were used, showing that these sterols had comparable effects regarding lipid bilayer order and complete solubilization of gel domains. However, confocal fluorescence microscopy of Giant Unilamellar Vesicles showed that zymosterol, a biosynthetic precursor of ergosterol and cholesterol, has no ability to form lipid rafts.In living cells, sterol-induced emission blue-shift and longer fluorescence lifetime of di-4-ANEPPS increased as: wild type<erg6Δ<CHO. Concomitantly, in model systems, cholesterol and zymosterol had much larger effects than ergosterol in di-4-ANEPPS fluorescence. However, zymosterol was unique because it yielded overlapping results for both saturated and unsaturated lipids. The two other sterols produced much stronger alterations with a saturated lipid, even though the absolute effect in di-4-ANEPPS fluorescence was often smaller than that of zymosterol. Hence, the differential regarding the interaction of sterols with saturated versus unsaturated lipids emerges as a requisite for lipid raft formation and as an important factor in sterol evolution.(1) F. Aresta-Branco, A.M. Cordeiro, H.S. Marinho, L. Cyrne, F. Antunes, R.F.M. de Almeida (2011) “Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, sphingolipid-enriched lipid rafts.” J. Biol. Chem. 286:5043-5054.Acknowledgements: PTDC/QUI-BIQ/104311/2008 and Ciencia2007 (FCT, Portugal).