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. 2007 Sep 1;93(5):1639-50.
doi: 10.1529/biophysj.107.107714. Epub 2007 May 11.

Formation of ceramide/sphingomyelin gel domains in the presence of an unsaturated phospholipid: a quantitative multiprobe approach

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

Formation of ceramide/sphingomyelin gel domains in the presence of an unsaturated phospholipid: a quantitative multiprobe approach

Bruno M Castro et al. Biophys J. .

Abstract

To better understand how ceramide modulates the biophysical properties of the membrane, the interactions between palmitoyl-ceramide (PCer) and palmitoyl-sphingomyelin (PSM) were studied in the presence of the fluid phospholipid palmitoyl-oleoyl-phosphatidylcholine (POPC) in membrane model systems. The use of two fluorescent membrane probes distinctly sensitive to lipid phases allowed a thorough biophysical characterization of the ternary system. In these mixtures, PCer recruits POPC and PSM in the fluid phase to form extremely ordered and compact gel domains. Gel domain formation by low PCer mol fraction (up to 12 mol %) is enhanced by physiological PSM levels (approximately 20-30 mol % total lipid). For higher PSM content, a three-phase situation, consisting of fluid (POPC-rich)/gel (PSM-rich)/gel (PCer-rich) coexistence, is clearly shown. To determine the fraction of each phase a quantitative method was developed. This allowed establishing the complete ternary phase diagram, which helps to predict PCer-rich gel domain formation and explains its enhancement through PSM/PCer interactions.

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Figures

FIGURE 1
FIGURE 1
Determination of t-PnA partition coefficient (Kp) between PSM-rich gel and POPC-rich fluid phase in POPC/PSM binary mixtures. The steady-state fluorescence anisotropy of t-PnA is represented as a function of the gel phase fraction in POPC/PSM mixtures. The gel phase mol fraction (Xg) was taken from the tie-line at 24°C of the POPC/PSM binary phase diagram (26). The line represents the nonlinear fit of Eq. 5 to the experimental data with formula image
FIGURE 2
FIGURE 2
Effect of PCer in POPC/PSM mixtures. (A) t-PnA and (B) DPH steady-state fluorescence anisotropy as a function of total sphingolipids mol fraction (XSL = XPSM + XPCer), in POPC/PSM mixtures containing 0 (○), 2 (□), 4 (⋄), 8 (▵), and 12 (*) mol % PCer. The dashed lines are merely a guide to the eye. The error bars are within the size of the symbol and correspond to at least three independent samples.
FIGURE 3
FIGURE 3
Formation of a gel phase that excludes DPH: effect of PCer on DPH steady-state fluorescence anisotropy in POPC/PSM mixtures containing (A) 0 (○) and 8 (▵) mol % PCer, and (B) 0 (○) and 12 (*) mol % PCer as a function of total sphingolipids molar fraction (XSL = XPSM + XPCer). The data present were taken from Fig. 2 B. The dashed lines are merely a guide to the eye. The error bars are within the size of the symbol and correspond to at least three independent samples.
FIGURE 4
FIGURE 4
Effect of PCer on t-PnA's mean fluorescence lifetime (〈τ〉) in POPC/PSM mixtures containing 0 (○), 2 (□), 4 (⋄), 8 (▵), and 12 (*) mol % PCer, as a function of total sphingolipids mol fraction (XSL = XPSM + XPCer). The dashed lines are merely a guide to the eye.
FIGURE 5
FIGURE 5
Fluorescence lifetime of the long component of t-PnA fluorescence decay, in POPC/PSM mixtures containing 0 (○), 2 (□), 4 (⋄), 8 (▵), and 12 (*) mol % PCer, as a function of total sphingolipids mol fraction (XSL = XPSM + XPCer). The dashed lines are merely a guide to the eye.
FIGURE 6
FIGURE 6
POPC/PSM/PCer ternary phase diagram at 24°C determined from the photophysical and phase-related properties of the two complementary fluorescent membrane probes t-PnA and DPH, together with quantification methodologies and an iterative procedure. (A) Ternary phase diagram highlighting the seven distinct regions identified. The abbreviations correspond to: F1, POPC-rich fluid phase; G1, PCer-rich gel, and G2, PSM-rich gel phase. The colors correspond to the number of phases present in each region: light gray, one phase; white, two phases; dark gray, three phases. (B) Scaled POPC/PSM/PCer phase diagram. The lines B1–B6 represent the phase boundaries between one-phase and two-phase regions. Each side of the tie-triangle is a phase boundary that separates a two-phase region from a three-phase region. T1–T4 are the tie-lines for the F1 + G1 region that contain the ternary mixtures composed of XSL = 0.2 and 2, 4, 8, and 12 mol % PCer (solid light gray circles), respectively. The open circles over B1 and B6 are the edges of the tie-lines. The direction and length of these tie-lines, together with points B and F allowed for the determination of B6 (see text for further details). The points A, B and C, D are experimental points taken from the POPC/PCer (23) and the POPC/PSM (26) phase diagrams, respectively. H and I are illustrative for which three-phase coexistence was quantified (Table 3). The lever rule was exemplified for these points (Appendix II). J and the dashed line FJ are presented for a better exemplification of the lever rule. The full lines were experimentally determined. All the lines have strong experimental basis and are thermodynamically consistent. However, due to technical limitations discussed in the text, B4 and B5 are still under definition, being represented by dashed lines. These boundaries are the best estimations based on our own experimental data, thermodynamic principles (42), and calorimetric studies (24). All the remainder of the phase diagram was determined independently of the exact position of B4 and B5.
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