The thermotropic behavior of palmitoylsphingomyelinvesicles containing GM1 gangliosideand cholesterol has been investigated by high-sensitivity differentialscanning calorimetry. The thermogramsexhibited by binary palmitoylsphingomyelin/GM1 mixtures are resolvableinto two components. Therelative contribution of the minor component, undetectable in theabsence of ganglioside, to the totalenthalpy and its transition temperature (>40
C) increase with theconcentration of the glycolipid embeddedin the vesicles. These data suggest the occurrence of lateralphase separation and that more ordered,higher melting GM1 ganglioside-enriched domains are present within thesphingomyelin bilayer. Studieson binary sphingomyelin/cholesterol mixtures confirmed the knowntendency of the sterol to decreasethe total enthalpy of sphingomyelin, forming cholesterol-enricheddomains. The thermograms exhibitedby ternary sphingomyelin/ganglioside/cholesterol mixtures in variableproportions (up to 20% molar GM1or Chol) displayed, on increasing the content of either the sterol orthe ganglioside, features addressableto sphingomyelin/cholesterol (peaks centered at temperature
40
C,decrease of enthalpy) or tosphingomyelin/GM1 mixtures (peaks centered at a temperature >40
C),respectively. This trend wasconfirmed by deconvolution analysis, showing that the thermograms areresolvable into componentsaddressable to GM1-enriched and to cholesterol-enriched domains.Taken all together, the results showthat the architectural features of sphingomyelin bilayers are stronglydependent on the presence of GM1ganglioside and cholesterol, whose presence is leading to the formationof separate, GM1-enriched andcholesterol-enriched distinct domains. Ganglioside-sphingomyelinand sphingomyelin-cholesterol,together with mutual ganglioside-ganglioside, interactions couldcontribute to maintain a network of bondsextending to proteins, forming specialized membrane domains, such ascaveolae, or others, whoseexperimental clues are the glycolipid-enriched detergent-insolublefractions that can be isolated from cellmembranes.