Copyright © by Biophysical Society. Final full-text version of the paper available at: http://www.biophysj.org/cgi/content/abstract/90/3/903
When cell membranes are treated with Triton X-100 or other detergents at 4 C, a nonsolubilized fraction can often
be recovered, the ‘‘detergent-resistant membranes’’, that is not found when detergent treatment takes place at 37 C. Detergentresistant
membranes may be related in some cases to membrane ‘‘rafts’’. However, several basic aspects of the formation of
detergent-resistant membranes are poorly understood. 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 Cer (5–30 mol % of Cer) 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 Cers do not give rise to detergent resistance. SM/Cer mixtures containing up to 30 mol % Cer
become fully soluble at ;50 C, i.e., well above the gel-fluid transition temperature of SM. The combined results of temperaturedependent
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 ;40 C). As a consequence, at temperatures allowing only partial solubilization,
the nonsolubilized 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 signaling, generation of ‘‘raft platforms’’, and detergent-resistant cell
membranes.
This work was supported in part by grants from the Spanish Ministerio
de Educación y Ciencia (No. BFU 2004-02955 to F.M.G., and No. BMC 2002-00784 to A.A.), and the University of the Basque Country
(UPV00042.310/13552 to F.M.G.). Research in the laboratory of LAB is
funded by a grant from the Danish Natural Science Research Council (SNF)
(21-03-0569) and the Danish National Research Foundation (which
supports MEMPHYS-Center for Biomembrane Physics). J.S. is a graduate
student supported by the Basque government.
Peer reviewed