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. 2005 Jun;88(6):4054-63.
doi: 10.1529/biophysj.104.054718. Epub 2005 Mar 25.

Domain formation and stability in complex lipid bilayers as reported by cholestatrienol

Y Jenny E Björkqvist  1 , Thomas K M Nyholm, J Peter Slotte, Bodil Ramstedt
Affiliations

Domain formation and stability in complex lipid bilayers as reported by cholestatrienol

Y Jenny E Björkqvist et al. Biophys J. 2005 Jun.

Abstract

In this study, we used cholestatrienol (CTL) as a fluorescent reporter molecule to study sterol-rich L(o) domains in complex lipid bilayers. CTL is a fluorescent cholesterol analog that mimics the behavior of cholesterol well. The ability of 12SLPC to quench the fluorescence of cholestatrienol gives a measure of the amount of sterol included in L(o) domains in mixed lipid membranes. The stability of sterol-rich domains formed in complex lipid mixtures containing saturated sphingomyelins, phosphatidylcholines, or galactosylceramide as potential domain-forming lipids were studied. The amount of sterol associated with sterol-rich domains seemed to always increase with increasing temperature. The quenching efficiency was highly dependent on the domain-forming lipid present in complex lipid mixtures. Sphingomyelins formed stable sterol-enriched domains and were able to shield CTL from quenching better than the other lipids included in this study. The saturated phosphatidylcholines also formed sterol-rich domains, but the quenching efficiency in membranes with these was higher than with sphingomyelins and the domains melted at lower temperatures. PGalCer was not able to form sterol-enriched domains. However, we found that PGalCer stabilized sterol-rich domains formed in PSM-containing bilayers. Using a fluorescent ceramide analog, we also demonstrated that N-palmitoyl-ceramide displaced the sterol from sphingolipid-rich domains in mixed bilayer membranes.

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Figures

FIGURE 1
FIGURE 1
Fluorescence emission intensity of CTL in phospholipid bilayers. (A). Samples consisted of phospholipids/cholesterol/CTL (95:4:1, molar ratio), and the intensity was measured at 25°C and 37°C. The figure shows the average ± range for duplicates of representative samples. (B). Fluorescence emission intensity of CTL in phospholipid bilayers as a function of temperature. The samples consisted of phospholipids/cholesterol/CTL (95:4:1, molar ratio) and were heated at a constant rate of 5°C/min.
FIGURE 2
FIGURE 2
Quenching of CTL emission by 12SLPC in phospholipid bilayers as a function of temperature. Emission intensities were measured in F and Fo samples, composed of POPC/(12SLPC or POPC)/phospholipids/cholesterol/CTL (30:30:30:9:1, molar ratio) heated by 5°C/min, and the F/Fo ratio was calculated. Temperature was increased by 5°C/min.
FIGURE 3
FIGURE 3
Domain formation in sphingolipid/cholesterol bilayers detected by 12SLPC quenching of CTL or tParGalCer as a function of temperature. (A). Fluorescence emission intensities were measured in F and Fo samples composed of POPC/(12SLPC or POPC)/sphingolipid/cholesterol/CTL (30:30:30:9:1, molar ratio), where the sphingolipid was PSM, PSM, and PGalCer (1:1) or PGalCer. (B). Fluorescence emission intensities were measured in F and Fo samples composed of POPC/(12SLPC or POPC)/sphingolipid/tParGalCer/cholesterol (30:30:29:1:10, molar ratio), where the sphingolipid was PSM and PGalCer (1:1) or PGalCer.
FIGURE 4
FIGURE 4
Representative thermograms of PSM/PGalCer bilayers with cholesterol. Equimolar mixtures of PSM and PGalCer containing 0–30 mol % cholesterol were heated and cooled at a rate of 0.5°C/min. Upscans are shown to the left, downscans to the right. Sterol concentrations are indicated in the figure.
FIGURE 5
FIGURE 5
Effect of ceramide on phospholipid/cholesterol domains detected by 12SLPC quenching of CTL and tParCer. F and Fo samples consisted of POPC/(12SLPC or POPC)/phospholipids/PCer/cholesterol (30:30:15:15:10, molar ratio). The effect was examined both with CTL (solid line) and tParCer (dashed line), which replaced 1 mol % of cholesterol or PCer, respectively.
FIGURE 6
FIGURE 6
Representative thermograms of equimolar mixtures of PCer and PSM. The samples were heated and cooled at a rate of 0.5°C/min.
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