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Background/Aims: Transient nanometric cholesterol- and sphingolipid-enriched domains, called rafts, are characterized by higher lipid order as compared to surrounding lipids. Here, we asked whether the seminal concept of highly ordered rafts could be refined with the presence of lipid domains exhibiting different enrichment in cholesterol and sphingomyelin and association with erythrocyte curvature areas. We also investigated how differences in lipid order between domains and surrounding membrane (bulk) are regulated and whether changes in order differences could participate to erythrocyte deformation and vesiculation. Methods: We used the fluorescent hydration- and membrane packing-sensitive probe Laurdan to determine by imaging mode the Generalized Polarization (GP) values of lipid domains vs the surrounding membrane. Results: Laurdan revealed the majority of sphingomyelin-enriched domains associated to low erythrocyte curvature areas and part of the cholesterol-enriched domains associated with high curvature. Both lipid domains were less ordered than the surrounding lipids in erythrocytes at resting state. Upon erythrocyte deformation (elliptocytes and stimulation of calcium exchanges) or membrane vesiculation (storage at 4°C), lipid domains became more ordered than the bulk. Upon aging and in membrane fragility diseases (spherocytosis), an increase in the difference of lipid order between domains and the surrounding lipids contributed to the initiation of domain vesiculation. Conclusion: The critical role of domain-bulk differential lipid order modulation for erythrocyte reshaping is discussed in relation with the pressure exerted by the cytoskeleton on the membrane.

Background/Aims: Transient nanometric cholesterol- and sphingolipid-enriched domains, called rafts, are characterized by higher lipid order as compared to surrounding lipids. Here, we asked whether the seminal concept of highly ordered rafts could be refined with the presence of lipid domains exhibiting different enrichment in cholesterol and sphingomyelin and association with erythrocyte curvature areas. We also investigated how differences in lipid order between domains and surrounding membrane (bulk) are regulated and whether changes in order differences could participate to erythrocyte deformation and vesiculation. Methods: We used the fluorescent hydration- and membrane packing-sensitive probe Laurdan to determine by imaging mode the Generalized Polarization (GP) values of lipid domains vs the surrounding membrane. Results: Laurdan revealed the majority of sphingomyelinenriched domains associated to low erythrocyte curvature areas and part of the cholesterolenriched domains associated with high curvature. Both lipid domains were less ordered than the surrounding lipids in erythrocytes at resting state. Upon erythrocyte deformation (elliptocytes and stimulation of calcium exchanges) or membrane vesiculation (storage at 4°C), lipid domains became more ordered than the bulk. Upon aging and in membrane fragility diseases (spherocytosis), an increase in the difference of lipid order between domains and the surrounding lipids contributed to the initiation of domain vesiculation. Conclusion: The critical role of domain-bulk differential lipid order modulation for erythrocyte reshaping is discussed in relation with the pressure exerted by the cytoskeleton on the membrane. [ABSTRACT FROM AUTHOR]

We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous SM, upon binding of a SM-specific nontoxic (NT) fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry [construct named His-mCherry-NT-lysenin (lysenin*)]. Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of 125I-lysenin* binding to erythrocytes upon SM depletion by SMase. The 125I-lysenin* binding isotherm indicated saturation at 3.5 × 106 molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled sub­micrometric domains on the plasma membrane of partially spread erythrocytes, colocalizing with inserted green BODIPY-SM, and abrogated by SMase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning, and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in RBCs gently suspended in 3D-gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous SM at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo.

We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous SM, upon binding of a SM-specific nontoxic (NT) fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry [construct named His-mCherry-NT-lysenin (lysenin*)]. Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of (125)I-lysenin* binding to erythrocytes upon SM depletion by SMase. The (125)I-lysenin* binding isotherm indicated saturation at 3.5 × 10(6) molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled sub-micrometric domains on the plasma membrane of partially spread erythrocytes, colocalizing with inserted green BODIPY-SM, and abrogated by SMase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning, and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in RBCs gently suspended in 3D-gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous SM at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)