Cholesterol Forms Submicrometric Domains On The Living Erythrocyte Membrane

Cholesterol is a major regulator of plasma membrane fluidity and deformability but its lateral distribution is poorly understood due to lack of suitable vital probes. The objective of our study was to address the level and stability of lateral cholesterol organization at the plasma membrane of living human erythrocyte. We here report the use of a non-toxic fragment of Clostridium perfringens toxin fused to the fluorescent monomeric mCherry protein (construct named theta*) to label endogenous plasma membrane cholesterol. Binding specificity of theta* was verified by retention on composition-defined liposomes and by measurement of 125I-labeled theta* binding to erythrocytes upon cholesterol depletion. By confocal microscopy, theta* revealed cholesterol submicrometric domains over another cholesterol pool, both in freshly spread but also in gel-suspended non-stretched erythrocytes, strongly supporting their relevance in vivo. Mechanistically, cholesterol domains on spread erythrocytes were stable in time and space, restricted by membrane anchorage to spectrin cytoskeleton via 4.1R complexes and temperature- and sphingomyelin-dependent, indicating regulation by membrane:cytoskeleton interaction and by intrinsic lipid packing. In conclusion, we show that theta* is a sensitive and quantitative probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells. This work was supported by grants from UCL (FSR), the F.R.S-FNRS, Salus Sanguinis foundation, ARC, IUAP and the Région Wallonne.