Rapid transbilayer movement of ceramides in phospholipid vesicles and in human erythrocytes.
The transbilayer diffusion of unlabeled ceramides with different acyl chains (C6-Cer, C10-Cer, and C16-Cer) was investigated in giant unilamellar vesicles (GUVs) and in human erythrocytes. Incorporation of a very small percentage of ceramides (approximately 0.1% of total lipids) to the external leaflet of egg phosphatidylcholine GUVs suffices to trigger a shape change from prolate to pear shape vesicle. By observing the reversibility of this shape change the transmembrane diffusion of lipids was inferred. We found a half-time for unlabeled ceramide flip-flop below 1 min at 37 degrees C. The rapid diffusion of ceramides in a phosphatidylcholine bilayer was confirmed by flip-flop experiments with a spin-labeled ceramide analogue incorporated into large unilamellar vesicles. Shape change experiments were also carried out with human erythrocytes to determine the trans-membrane diffusion of unlabeled ceramides into a biological membrane. Addition of exogenous ceramides to the external leaflet of human erythrocytes did not trigger echinocyte formation immediately as one would anticipate from an asymmetrical accumulation of new amphiphiles in the outer leaflet but only after approximately 15 min of incubation at 20 degrees C in the presence of an excess of ceramide. We interpret these data as being indicative of a rapid ceramide equilibration between both erythrocyte leaflets as indicated also by electron spin resonance spectroscopy with a spin-labeled ceramide. The late appearance of echinocytes could reveal a progressive trapping of a fraction of the ceramide molecules in the outer erythrocytes leaflet. Thus, we cannot exclude the trapping of ceramides into plasma membrane domains.
López-Montero
Iván
I
Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie 75005 Paris, France.
Rodriguez
Nicolas
N
Cribier
Sophie
S
Pohl
Antje
A
Vélez
Marisela
M
Devaux
Philippe F
PF
D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy; D004907:Erythrocyte Deformability / Q000502:physiology; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D010743:Phospholipids / Q000378:metabolism; D013113:Spin Labels; D013696:Temperature
D002518:Ceramides; D008051:Lipid Bilayers; D010743:Phospholipids; D013113:Spin Labels