Detergent-resistant, ceramide-enriched domains in sphingomyelin/ceramide bilayers

¹ Unidad de Biofísica
² University of Southern Denmark
³ Universidad del Pais Vasco Departamento de Bioquimica
⁴ Universidad del Pais Vasco Unidad de Biofisica

^* To whom correspondence should be addressed. E-mail: gbpaliza{at}lg.ehu.es.

Submitted on June 3, 2005
Revised on August 9, 2005
Accepted on 11 October 2005

	Abstract

When cell membranes are treated with Triton X-100 or other detergents at 4° C, a non-solubilized fraction can often be recovered, the "detergent-resistant membranes", that is not found when detergent treatment takes place at 37° C. Detergent-resistant membranes may be related in some cases to membrane "rafts". However, several basic aspects of the formation of detergent resistant membranes are poorly understood. In order to answer some of the relevant questions, a simple bilayer composition that would mimic detergent-resistant membranes was required. The screening of multiple lipid compositions has shown that the binary mixture egg sphingomyelin/egg ceramide (SM/Cer) exhibits the required detergent resistance. In detergent-free membranes composed of different mixtures of SM and ceramide (5 to 30 mol% of ceramide) differential scanning calorimetry, fluorescence spectroscopy and fluorescence microscopy experiments reveal the presence of discrete, Cer-enriched, gel domains in a broad temperature range. In particular, at temperatures below SM phase transition (40° C) two gel (respectively Cer-rich and SM-rich) phases are directly observed using fluorescence microscopy. Although pure SM membranes are fully solubilized by Triton X-100 at room temperature, 5 mol % Cer is also enough to induce detergent resistance, even with a large detergent excess and lengthy equilibration times. Short-chain ceramides do not give rise to detergent resistance. SM/Cer mixtures containing up to 30 mol % Cer become fully soluble at ca. 50° C, i.e. well above the gel-fluid transition temperature of SM. The combined results of temperature-dependent solubilization and differential scanning calorimetry reveal that SM-rich domains are preferentially solubilized over the Cer-rich ones as soon as the former melt (i.e. at ca. 40° C). As a consequence, at temperatures allowing only partial solubilization, the non-solubilized residue is enriched in Cer with respect to the original bilayer composition. Fluorescence microscopy of giant unilamellar vesicles at room temperature clearly shows that SM-rich domains are preferentially solubilized over the Cer-rich ones, and that the latter become more rigid and extensive as a consequence of the detergent effects. These observations may be relevant to the phenomena of sphingomyelinase-dependent signalling, generation of "raft platforms", and detergent-resistant cell membranes.

Key Words: Ceramide, Detergent-resistant membranes, Membrane, Membrane domains, Sphingomyelin/ceramide bilayers