Biophysical properties of ergosterol-enriched lipid rafts in yeast and tools for their study: characterization of ergosterol/phosphatidylcholine membranes with three fluorescent membrane probes

• Published in: Chemistry and physics of lipids Vol. 165; no. 5; pp. 577 - 588
• Main Authors: Bastos, André E.P., Marinho, H. Susana, Cordeiro, André M., de Soure, António M., de Almeida, Rodrigo F.M.
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier Ireland Ltd 01-07-2012
• Subjects: Biophysical Phenomena; Ergosterol; Ergosterol - chemistry; Ergosterol - metabolism; Fluorescence; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; gels; Head-labeled phospholipids; hydrogen bonding; Liposomes; Membrane Microdomains - metabolism; phosphatidylcholines; Phosphatidylcholines - chemistry; Phosphatidylcholines - metabolism; Potential sensitive dyes; Saccharomyces cerevisiae - cytology; Spectrometry, Fluorescence; Sterol–lipid interactions; yeasts; Ergosterol; Chol; lo; NBD-DPPE; Fluorescence; FLIM; Potential sensitive dyes; LUV; TMA-DPH; Homo-FRET; t-PnA; PC; di-4-ANEPPS; MLV; POPC; Sterol–lipid interactions; ld; Tm; Rhod-DOPE; Erg; Liposomes; DPH; Head-labeled phospholipids; DPPC
• ISSN: 0009-3084; 1873-2941
• DOI: 10.1016/j.chemphyslip.2012.06.002

► Three fluorescent membrane probes were used to study ergosterol-containing membranes. ► Fluorescent probes reflect the hydration behavior of ergosterol/phospholipid mixtures. ► Di-4-ANEPPS fluorescence properties are sensitive to sterol type and content. ► Di-4-ANEPPS fluorescence lifetime responds uniquely to sterol–lipid interactions. ► Several probe applications in the study of yeast membrane rafts are presented.
In this work, binary mixtures of phospholipid/ergosterol (erg) were studied using three fluorescent membrane probes. The phospholipid was either saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) or monounsaturated (1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphocholine, POPC) phosphatidylcholine, to evaluate the fluorescence properties of the probes in gel, liquid ordered (lo) and liquid disordered (ld) phases. The probes have been used previously to study cholesterol-enriched domains, but their photophysical properties in erg-enriched membranes have not been characterized.
N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DPPE) presents modest blue-shifts upon erg addition, and the changes in the fluorescence lifetime are mainly due to differences in the efficiency of its fluorescence dynamic self-quenching. However, the steady-state fluorescence anisotropy of NBD-DPPE presents well-defined values in each lipid phase.
N-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Rhod-DOPE) presents a close to random distribution in erg-rich membranes. There are no appreciable spectral shifts and the steady-state fluorescence anisotropy presents complex behavior, as a result of different photophysical processes. The probe is mostly useful to label ld domains in yeast membranes.
4-(2-(6-(Dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium (di-4-ANEPPS) is an electrochromic dye with excitation spectra largely insensitive to the presence of erg, but presenting a strong blue-shift of its emission with increasing concentrations of this sterol. Its partition coefficient is favorable to lo domains in POPC/erg mixtures. Although the fluorescence properties of di-4-ANEPPS are less sensitive to erg than to chol, in both cases the fluorescence lifetime responds monotonically to sterol mole fraction, becoming significantly longer in the presence of sterol as compared to pure POPC or DPPC bilayers. The probe displays a unique sensitivity to sterol–lipid interaction due to the influence of hydration and H-bonding patterns at the membrane/water interface on its fluorescence properties. This makes di-4-ANEPPS (and possibly similar probes) potentially useful in the study of erg-enriched domains in more complex lipid mixtures and in the membranes of living yeast cells.