Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation

Abstract

Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.

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

“…Statistical significance was tested either with two-sample t-test or one-way ANOVA followed by Tukey's post-hoc test (NS, not significant; * p < 0.05; ** p < 0.01 and *** p < 0.001). [25,26] and preferentially associate with high and low curvature areas of the biconcave RBC membrane [29]. This analysis was limited to chol and SM while not considering PC, another abundant class of outer PM leaflet lipids, or glycosphingolipids, known to play key pathophysiological roles.…”

“…Lysenin* and theta* were produced as previously described [25,26], dissolved in 1 mg/ml DMEM-BSA (Bovine Serum Albumin, Sigma) and cleared of aggregates before each experiment by centrifugation at 20, 000 g for 10 min. RBC labeling with toxins* was performed in suspension (i.e.…”

“…Chol content was determined as previously described [25]. SM content modulation was achieved with sphingomyelinase from Bacillus Cereus (Sigma-Aldrich) as described in [26].…”

“…super resolution microscopy or fluorescence lifetime spectroscopy) [13][14][15][16] and more relevant lipid probes [17,18] have allowed to evidence bigger (submicrometric instead of nanometric) and more stable lipid domains. Those have been observed on prokaryotic cells [19], yeast [20,21] and various eukaryotic cells like keratinocytes [22], fibroblasts [23] and RBCs [24][25][26]. Focus is generally made on sphingolipids and sterols as they are two major lipids of the PM external leaflet of many cells and because they are known to be enriched in rafts and associated with membrane fluidity regulation.…”

“…To label the domains for fluorescence microscopy, we used fluorescent lipid analogs (i.e. BODIPY-SM) that get inserted at trace levels in the PM [27] and developed and carefully validated fluorescent toxin fragments specific to endogenous chol (theta*) and SM (lysenin*) [25,26]. More recently, we also used atomic force microscopy, a high-resolution technique applicable to cells in their native state (i.e.…”