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Use of Cyclodextrin for AFM Monitoring of Model Raft Formation

Centre de Biochimie Structurale, CNRS UMR 5048-Université Montpellier I, INSERM UMR554, 34090 Montpellier Cedex, France

Correspondence: Address reprint requests to Christian Le Grimellec, C.B.S., INSERM UMR554 29, rue de Navacelles, 34090 Montpellier Cedex, France. Tel.: 33-467-41-79-07; Fax: 33-467-41-79-13; E-mail: clg{at}cbs.univ-montp1.fr.

The lipid rafts membrane microdomains, enriched in sphingolipids and cholesterol, are implicated in numerous functions of biological membranes. Using atomic force microscopy, we have examined the effects of cholesterol-loaded methyl-ß-cyclodextrin (MßCD-Chl) addition to liquid disordered (l_d)-gel phase separated dioleoylphosphatidylcholine (DOPC)/sphingomyelin (SM) and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SM supported bilayers. We observed that incubation with MßCD-Chl led to the disappearance of domains with the formation of a homogeneously flat bilayer, most likely in the liquid-ordered (l_o) state. However, intermediate stages differed with the passage through the coexistence of l_o-l_d phases for DOPC/SM samples and of l_o-gel phases for POPC/SM bilayers. Thus, gel phase SM domains surrounded by a l_o matrix rich in cholesterol and POPC could be observed just before reaching the uniform l_o state. This suggests that raft formation in biological membranes could occur not only via liquid-liquid but also via gel-liquid immiscibility. The data also demonstrate that MßCD-Chl as well as the unloaded cyclodextrin MßCD make holes and preferentially extract SM in supported bilayers. This strongly suggests that interpretation of MßCD and MßCD-Chl effects on cell membranes only in terms of cholesterol movements have to be treated with caution.

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