Tuning of Differential Lipid Order Between Submicrometric Domains and Surrounding Membrane Upon Erythrocyte Reshaping

Abstract

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.

Spatial Relationship and Functional Relevance of Three Lipid Domain Populations at the Erythrocyte Surface

“…2B) while only a part of chol-enriched domains in HC was labeled (red arrows, Fig. 2B), in agreement with our previous study [31]. Thanks to the Laurdan fluorescence emission at two wavelengths, we next calculated the Generalized Polarization (GP, see material and methods; proportional to the membrane lipid order) of the chol-, SM-and PC-enriched domains.…”

“…As previously observed, chol-enriched domains located in HC exhibited a higher lipid order than those present in LC areas (compare stripped to full red columns, Fig. 2C) [31]. On the other hand, all the polar lipid-enriched domains in LC, including those enriched in PC, exhibited a lower lipid order than the surrounding membrane (compare colored columns to the grey column, Fig.…”

“…lipid order); (ii) the strength of their cytoskeleton anchorage; and/or (iii) their correspondence with specific lipids in the inner leaflet, a.o.. The membrane lipid order hypothesis is supported by (i) our present observation that SM/PC/chol-enriched domains were more disordered than the rest of the membrane; (ii) our previous observation that lipid domain order increases to a bigger extent than the bulk membrane order upon stimulation of calcium efflux by treatment of RBCs with EGTA [31]; and (iii) several studies in model membranes, although with sometimes conflicting information. For instance, PMCA activity is decreased in highly-ordered areas of liposomes made of PC/SM/chol [64] but is favored in highly ordered lens fiber lipids in comparison with disordered DOPC liposomes [65].…”

“…RBCs were then immobilized and examined using (i) confocal mode for lipids and (ii) multiphoton mode with acquisition at 440 nm and 490 nm and a Normaski prism for Laurdan. Lipid order determination was performed as described in [30] and in [31]. Briefly, domains and surrounding membrane ROIs and masks were obtained from the two fluorescent channels (440 nm and 490 nm) of Laurdan images.…”

Plasma Membrane Lipid Domains as Platforms for Vesicle Biogenesis and Shedding?

“…Second, giant PM vesicles (GPMVs) derived from living cells [ 199 ] reveal that L o and L d phases in natural PM can assume a wide range of lipid order states [ 215 ]. Finally, regions of different lipid order than the bulk have been shown at the surface of several cells including RBCs, platelets and monocytes [ 216 , 217 , 218 , 219 , 220 ] (see below).…”