Use of cyclodextrin for AFM monitoring of model raft formation.
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-beta-cyclodextrin (MbetaCD-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 MbetaCD-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 MbetaCD-Chl as well as the unloaded cyclodextrin MbetaCD make holes and preferentially extract SM in supported bilayers. This strongly suggests that interpretation of MbetaCD and MbetaCD-Chl effects on cell membranes only in terms of cholesterol movements have to be treated with caution.
Giocondi
Marie-Cécile
MC
Centre de Biochimie Structurale, CNRS UMR 5048-Université Montpellier I, INSERM UMR554, 34090 Montpellier Cedex, France.
Milhiet
Pierre Emmanuel
PE
Dosset
Patrice
P
Le Grimellec
Christian
C
D002784:Cholesterol / Q000737:chemistry*; D003505:Cyclodextrins / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D046911:Macromolecular Substances; D008560:Membrane Fluidity; D021962:Membrane Microdomains / Q000737:chemistry* / Q000000981:diagnostic imaging*; D018625:Microscopy, Atomic Force / Q000379:methods*; D008968:Molecular Conformation; D010539:Permeability; D044367:Phase Transition; D010713:Phosphatidylcholines / Q000737:chemistry; D013109:Sphingomyelins / Q000737:chemistry; D014463:Ultrasonography
D003505:Cyclodextrins; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine