Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane

Abstract

Supplementary key words toxin • His-mCherry-NT-lysenin • lateral membrane heterogeneity • vital confocal imaging • membrane tension • cholesterol • temperatureLipids at the outer leafl et of the mammalian plasma membrane are mainly composed of: i ) SM, the most abundant sphingolipid (SL), based on a ceramide backbone and bearing a phosphocholine polar head; ii ) glycosphingolipids (GSLs), another group of SLs bearing various sugars instead of phosphocholine, from simple glucosylceramide (GlcCer) to complex GSLs such as GM1 [for a review, see ( 1 )]; iii ) phosphatidylcholine (PC), the major glycerophospholipid, sharing the same phosphocholine polar head as SM; and iv ) nonpolar cholesterol. Lipid bilayers are no longer considered as a homogenous solvent for membrane proteins ( 2 ), but are now represented with lateral heterogeneity at two different scales of time and space: i ) transient nanometric "lipid rafts", defi ned as small clusters enriched in SLs, sterol, and GPI-anchored proteins ( 3, 4 ); versus ii ) submicrometric/mesoscale domains ( 5-15 ). These larger and more stable domains are well-characterized on artifi cial vesicles ( 16, 17 ), but their relevance for living cells has been questioned ( 18,19 ).The occurrence in living cells of submicrometric/mesoscale domains was fi rst inferred from unexpected behavior in fl uorescence recovery after photobleaching (FRAP)

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.…”

“…Chol content was determined as previously described [25]. SM content modulation was achieved with sphingomyelinase from Bacillus Cereus (Sigma-Aldrich) as described in [26]. To inhibit mechano-activated channels, labeled and immobilized RBCs were incubated with 7 µM GsMTx4 peptide (Abcam) for 15 min and observed upon treatment maintenance.…”

“…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.…”

“…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.…”