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Biophys. J. BioFAST: First Published March 24, 2006. doi:10.1529/biophysj.105.077107
© 2006 by the Biophysical Society.

A more recent version of this article appeared on June 15, 2006.
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MEMBRANES

Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol

Matthias Fidorra 1, Lars Duelund 2, Chad Leidy 3, Adam Simonsen 4 and Luis A. Bagatolli 5*

1 MEMPHYS - Center for Biomembrane Physics/Dept. of Physics, University of Southern Denmark
2 MEMPHYS - Center for Biomembrane Physics/Dept of Chemistry, University of Southern Denmark
3 Department of Physics,Universidad de los Andes, Colombia
4 MEMPHYS - Center for Biomembrane Physics/Dept of Physics, University of Southern Denmark
5 MEMPHYS /Department of Biochemistry and Molecular Biology, University of Southern Denmark

* To whom correspondence should be addressed. E-mail: bagatolli{at}memphys.sdu.dk.

Submitted on November 9, 2005
Revised on December 16, 2005
Accepted on 28 February 2006


  Abstract
The effect of temperature on the lateral structure of lipid bilayers composed of porcine brain ceramide and 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC), with and without addition of cholesterol, were studied using differential scanning calorimetry (DSC), Fourier transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM) and confocal/two-photon excitation fluorescence microscopy (which include LAURDAN generalized polarization function images). A broad gel/fluid phase coexistence temperature regime, characterized by the presence of micrometer sized gel-phase domains with stripe and flower-like shapes, was observed for different POPC/ceramide mixtures (up to ~25 mol % ceramide). This observed phase coexistence scenario is in contrast to that reported previously for this mixture (Biochim Biophys Acta, 2001, 1510:167-184) where absence of gel/fluid phase coexistence was claimed using bulk LAURDAN generalized polarization (GP) measurements. We demonstrate that this apparent discrepancy (based in the direct comparison between the LAURDAN GP data obtained in the microscope and the fluorometer) disappears when the additive property of the LAURDAN GP function is taken into account to examine the data obtained using bulk fluorescence measurements. Addition of cholesterol to the POPC/ceramide mixtures shows a gradual transition from a gel/fluid to gel/liquid-ordered phase coexistence scenario as indicated by the different experimental techniques used in our experiments. This last results suggest the absence of fluid-ordered/fluid-disordered phase coexistence in the ternary mixtures studied in contrast to that observed at similar molar concentrations with other ceramide-base containing lipid mixtures (such as POPC/sphingomyelin/cholesterol, which is used as a canonical raft model membrane). Additionally, we observe a critical cholesterol concentration in the ternary mixtures that generates a peculiar lateral pattern characterized by the observation of three distinct regions in the membrane.

Key Words: LAURDAN, POPC, ceramide, cholesterol, giant vesicles, lipid domains




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Copyright © 2006 by the Biophysical Society.