Raft-like domain formation in large unilamellar vesicles probed by the fluorescent phospholipid analogue, C12NBD-PC.
The liquid-ordered/disordered-phase domain co-existence in large unilamellar vesicle membranes consisting of phosphatidylcholine:sphingomyelin (2:1) with different amounts of cholesterol has been examined using a concentration-dependent self-quenching of a single reporter molecule, C12NBD-PC. A temperature-dependent decrease of fluorescence intensity was associated with the expected formation and increase of l(o)-phase membrane fraction in the vesicles. The result is consistent with exclusion of the fluorescent probe from the liquid-ordered phase which partitions preferentially into the liquid-disordered phase membrane domains. This leads to an increase of the local concentration of fluorophore in the liquid-disordered phase and a decrease of the quantum yield. This effect was used to obtain a quantitative estimation of the fraction of the vesicle membrane occupied by the liquid-ordered phase, Phi(o), as a function of temperature and cholesterol content between 0 and 45 mol%. The value of Phi(o) was related to the assumed partition coefficient k(p) of probe between liquid-ordered/disordered phases. For large unilamellar vesicles containing 20 and 4 mol% cholesterol and probe, respectively, with k(p) = 0 (probe completely excluded from liquid-ordered phase), Phi(o) = 0.16 and with k(p) = 0.2, Phi(o) = 0.2. The results are relevant to the action of detergent in the fractionation of detergent-resistant membrane from living cells.
Coste
Virginie
V
Université Pierre et Marie Curie - Paris 6, UMR INSERM 538, CHU St Antoine, 27 rue Chaligny, Paris F-75012, France.
Puff
Nicolas
N
Lockau
Daniel
D
Quinn
Peter J
PJ
Angelova
Miglena I
MI
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D004530:Egg Yolk; D005456:Fluorescent Dyes; D008081:Liposomes; D021962:Membrane Microdomains / Q000502:physiology* / Q000648:ultrastructure*; D018715:Microscopy, Video; D010713:Phosphatidylcholines; D010743:Phospholipids; D013816:Thermodynamics
D005456:Fluorescent Dyes; D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; C067460:1-oleoyl-2-(12-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D009327:4-Chloro-7-nitrobenzofurazan