Detergent-Resistant, Ceramide-Enriched Domains in Sphingomyelin/Ceramide Bilayers

Abstract

When cell membranes are treated with Triton X-100 or other detergents at 4 degrees C, a nonsolubilized fraction can often be recovered, the "detergent-resistant membranes", that is not found when detergent treatment takes place at 37 degrees 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. 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 ( approximately 40 degrees 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 approximately 50 degrees 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 approximately 40 degrees 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.

Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers

“…The importance of ceramide in modulating membrane physical properties and biological function has prompted a number of studies of ceramide incorporation in model membranes. Several recent investigations have examined the effects of ceramide incorporation on coexisting ordered and fluid domains in phase separated binary and ternary lipid mixtures that are used to model membrane rafts Fidorra et al, 2006;Silva et al, 2007;Sot et al, 2006Sot et al, , 2008. This work has demonstrated that ceramide leads to increased membrane heterogeneity and that its effects depend on the membrane composition.…”

The many faces (and phases) of ceramide and sphingomyelin II – binary mixtures

“…The LC (16:0 Cer-enriched) phase observed in monolayers at X 16:0Cer > 0.4 is, in fact, a mixture of two condensed phases (Busto et al 2009). Also, Sot et al (2006) observed domain coexistence over a large region of the egg SM/egg Cer temperature-composition diagram by using calorimetric, fluorescence quenching, and temperaturedependent turbidimetric measurements.…”

“…Cer-induced domain segregation in several lipid mixtures with glycerophospholipids (Huang et al 1996;Carrer and Maggio 1999;Veiga et al 1999;Fanani et al 2002;Hsueh et al 2002;Sot et al 2005Sot et al , 2006Carrer et al 2006) has been reported using different biophysical approaches [see Alonso (2006, 2009) for reviews]. Cers induce high-temperature melting domains in mixtures where phospholipids display low (palmitoyl-oleoyl-PC) (Hsueh et al 2002) and high (dipalmitoyl-PC or dielaidoylphosphatidylethanolamine) (Carrer and Maggio 1999;Sot et al 2005) gel-to-fluid phase transition temperatures.…”

“…Cers induce high-temperature melting domains in mixtures where phospholipids display low (palmitoyl-oleoyl-PC) (Hsueh et al 2002) and high (dipalmitoyl-PC or dielaidoylphosphatidylethanolamine) (Carrer and Maggio 1999;Sot et al 2005) gel-to-fluid phase transition temperatures. A microscopy study combining DSC, fluorescence spectroscopy, and confocal fluorescence on the interaction of egg Cer with its relative egg SM showed the segregation of detergentresistant Cer-enriched domains (Sot et al 2006). In either premixed or enzyme-generated systems with bovine brain SM [mainly stearic (18:0) and lignoceric (24:0) acyl chains], Cer induces morphologically different Cer-enriched domains, depending on how the mixture was generated (Fanani et al 2002De Tullio et al 2008).…”

“…In both systems, 16:0 Cer exerts a number of effects in a composition-dependent manner up to X 16:0 Cer 0.3-0.4 (Busto et al 2009). An interpretation of the data on the basis of lipid bilayer observations (Sot et al 2006) is that 16:0 Cerrich and 16:0 Cer-poor phases are immiscible and phase-segregated. In the range X 16:0 Cer 0-0.4, monolayer experiments show a strong condensing effect exerted by the addition of 16:0 Cer (Fig.…”