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. 2007 Jan 15;92(2):502-16.
doi: 10.1529/biophysj.106.091876. Epub 2006 Oct 20.

Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid

Liana C Silva  1 , Rodrigo F M de Almeida, Bruno M Castro, Alexander Fedorov, Manuel Prieto
Affiliations

Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid

Liana C Silva et al. Biophys J. .

Abstract

The effect of physiologically relevant ceramide concentrations (< or = 4 mol %) in raft model membranes with a lipid composition resembling that of cell membranes, i.e., composed of different molar ratios of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol (Chol) along a liquid-disordered-liquid-ordered tie line was explored. The application of a fluorescence multiprobe and multiparameter approach, together with multiple fluorescence resonance energy transfer (FRET) pairs, in the well-characterized palmitoyl-oleoyl-phosphocholine (POPC)/palmitoyl-sphingomyelin (PSM)/Chol ternary mixture, revealed that low palmitoyl-ceramide (PCer) concentrations strongly changed both the biophysical properties and lipid lateral organization of the ternary mixtures in the low-to-intermediate Chol/PSM-, small raft size range (<25 mol % Chol). For these mixtures, PCer recruited up to three PSM molecules for the formation of very small ( approximately 4 nm) and highly ordered gel domains, which became surrounded by rafts (liquid-ordered phase) when Chol/PSM content increased. However, the size of these rafts did not change, showing that PCer did not induce the formation of large platforms or the coalescence of small rafts. In the high Chol/PSM-, large raft domains range (>33 mol % Chol), Chol completely abolished the effect of PCer by competing for PSM association. Lipid rafts govern the biophysical properties and lateral organization in these last mixtures.

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Figures

FIGURE 1
FIGURE 1
Ternary phase diagram of POPC/PSM/Chol mixtures (adapted from de Almeida et al. (24)). The black line corresponds to the tie line that contains the 1:1:1 POPC/PSM/Chol mixture. The black dots in the tie line define this composition and the extremes of the tie line. The POPC/PSM/Chol ratios for the mixtures used in this study were taken from this tie line and are represented by the white dots. The gray shades represent regions of different sized raft domains: in the low-to-intermediate Chol/PSM range (light gray) raft domains are small (ranging from <20 to ∼75 nm), whereas in the high Chol/PSM range (dark gray) larger rafts are formed (>∼100 nm).
FIGURE 2
FIGURE 2
Effect of low PCer amounts on the fluorescence anisotropy of the probes in the ternary mixtures. (A) t-PnA (solid symbols) and DPH (open symbols) anisotropy in ternary mixtures containing 0 (circles), 2 (diamonds), and 4 (squares) mol % PCer. (B) Anisotropy of Rho-DOPE (*,+), NBD-DPPE (−,▴), and NBD-DOPE (×,▵) for ternary mixtures containing 0 and 4 mol % PCer, respectively. The lines are merely a guide to the eye. The error bars are within the size of the symbol and correspond to at least five independent measurements.
FIGURE 3
FIGURE 3
Variation of the lifetime components of t-PnA fluorescence decay with lipid composition for the ternary POPC/PSM/Chol mixtures along the tie line that crosses the 1:1:1 mixture at room temperature containing (A) 0, (B) 2, and (C) 4 mol % PCer.
FIGURE 4
FIGURE 4
Effect of PCer on the fluorescence lifetime of the probes. (A) Mean fluorescence lifetime of t-PnA in the ternary mixtures containing 0 (circles), 2 (diamonds), and 4 (squares) mol % PCer. (B) Lifetime-weighted quantum yield of NBD-DPPE (−,▴) and NBD-DOPE (×,▵) in ternary mixtures containing 0 and 4 mol % PCer, respectively. The dotted lines are only a guide to the eye.
FIGURE 5
FIGURE 5
Variation of t-PnA fluorescence anisotropy with temperature for (A) POPC/PSM/Chol: 0.72:0.23:0.05 molar ratio, (B) POPC/PSM/Chol: 0.60:0.26:0.14 molar ratio, and (C) POPC/PSM/Chol: 0.25:0.35:0.40 molar ratio, containing 0 (circles), 2 (diamonds), and 4 (squares) mol % PCer. The dotted lines are only a guide to the eye. The Tm was taken by the midpoint of the intersection of the lines describing the initial (gel), intermediate (gel and fluid), and final (fluid) regimes (see de Almeida et al. (23) for additional details).
FIGURE 6
FIGURE 6
Variation of FRET efficiency, E, for the donor/acceptor pair (A) NBD-DPPE/Rho-DOPE, (B) t-PnA/NBD-DPPE, and (C) t-PnA/NBD-DOPE as a function of lo molar fraction, Xlo, in the lo + ld coexistence region along the tie line of the ternary phase diagram for the POPC/PSM/Chol mixture (24) containing 0 (circles) and 4 (squares) mol % PCer. Experimental data are represented by solid symbols, and open symbols are values from theoretical calculations. The open circles were obtained by calculation of the expected E for a random distribution of donors and acceptors. The open squares correspond to (A) the expected E in a situation where surface area for probe distribution decreases due to formation of gel domains that exclude both donor and acceptors, and (B,C) the expected E when donors located both in the gel and fluid phases are transferring energy to acceptors randomly distributed in the fluid phases. In these calculation XG = 15% and a Re = 3.8 and 4.0 nm for NBD-DPPE and NBD-DOPE, respectively, were assumed (see Discussion and Appendix for further details). The open triangle and the open diamond (B) correspond to the expected E for a situation where PCer/PSM gel domains are surrounded by only ld or lo phases, respectively. It was assumed that donors located in the gel phase were only able to transfer to one of the fluid phases, XG = 15%, Re = 3.8, and the amount of donor in the gel phase and the surface density of the acceptors in each fluid phase was taken into account (see Appendix for further details). The dotted and dotted-dashed lines are only to guide the eye. The vertical dashed lines correspond to the mixtures containing 25% and 33% Chol. Time-resolved FRET measurements are extremely reproducible, and variations in the values of E are within <1.5% for completely independent samples.
FIGURE 7
FIGURE 7
Schematic representation of the effect of PCer in lipid rafts biophysical properties and organization. (A) By recruiting PSM, PCer changes the composition of the mixtures and consequently the position of the tie line of the remaining fluid phase. The dashed black line and the white dots correspond to the estimated composition of the fluid phase that remains after the sequestering of PSM for gel-domain formation (see text for details). When increasing Chol content, the effect of PCer is opposed and less PSM is recruited for domain formation until 25%–33% Chol is reached and gel-domain formation completely abolished. Thus, the line that defines fluid phase composition should connect with the tie line in this region. According to this model, Xlo should decrease from 26% and 58% to 21% and 54%, respectively, when 4% PCer is present. (B) For the 100% ld phase one PCer molecule recruits up to three PSM molecules and forms highly ordered PCer/PSM-gel domains. The amount of gel formed is considerable, XG ∼ 15%, but the size of the domains is small, ∼4 nm (see Discussion). Panels B, C, and D are a pictorial top view of the bilayer because ∼250 molecules should be involved in the formation of a nanodomain of this dimension. (C) In the low-to-intermediate XChol, i.e., in the range of small sized rafts, PCer/PSM-gel domains are still present and are surrounded by lipid rafts (lo phase). FRET experiments show that PCer is not forming platforms or promoting the coalescence of the small rafts into large ones. (D) In the high Chol, large sized rafts range, PCer ability to form gel domains with PSM is abolished by the presence of Chol that competes for the association with PSM. In this situation, lipid rafts are governing membrane properties.
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