caa a22 4500 606177043 CHVBK 20210128100750.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00018-015-1951-x doi (NATIONALLICENCE)springer-10.1007/s00018-015-1951-x Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation [Elektronische Daten] [Mélanie Carquin, Louise Conrard, Hélène Pollet, Patrick Van Der Smissen, Antoine Cominelli, Maria Veiga-da-Cunha, Pierre Courtoy, Donatienne Tyteca] 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. Springer Basel, 2015 His-mCherry-theta-D4 nationallicence 125I-toxin nationallicence Lateral membrane heterogeneity nationallicence Vital confocal imaging nationallicence Membrane tension nationallicence Temperature nationallicence C2C12 myoblasts nationallicence BODIPY-sphingomyelin nationallicence CalA : Calyculin A nationallicence DF-BSA : Defatted-bovine serum albumin nationallicence FRAP : Fluorescence recovery after photobleaching nationallicence LatB : Latrunculin B nationallicence mβCD : Methyl-β-cyclodextrin nationallicence MLVs : Multilamellar vesicles nationallicence PC : Phosphatidylcholine nationallicence PKC : Protein kinase C nationallicence PMA : Phorbol 12-myristate 13-acetate nationallicence RBC : Red blood cell nationallicence SEM : Scanning electron microscopy nationallicence SM : Sphingomyelin nationallicence SMase : Sphingomyelinase nationallicence theta* : His-mCherry-theta-D4 nationallicence Carquin Mélanie CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut Conrard Louise CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut Pollet Hélène CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut Van Der Smissen Patrick CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut Cominelli Antoine CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut Veiga-da-Cunha Maria Laboratory of Physiological Chemistry, de Duve Institute and Université Catholique de Louvain, 1200, Brussels, Belgium aut Courtoy Pierre CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut Tyteca Donatienne CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut Cellular and Molecular Life Sciences Springer International Publishing 72/23(2015-12-01), 4633-4651 1420-682X 72:23<4633 2015 72 18 https://doi.org/10.1007/s00018-015-1951-x text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00018-015-1951-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Carquin Mélanie CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut NATIONALLICENCE 700 1- Conrard Louise CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut NATIONALLICENCE 700 1- Pollet Hélène CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut NATIONALLICENCE 700 1- Van Der Smissen Patrick CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut NATIONALLICENCE 700 1- Cominelli Antoine CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut NATIONALLICENCE 700 1- Veiga-da-Cunha Maria Laboratory of Physiological Chemistry, de Duve Institute and Université Catholique de Louvain, 1200, Brussels, Belgium aut NATIONALLICENCE 700 1- Courtoy Pierre CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut NATIONALLICENCE 700 1- Tyteca Donatienne CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium aut NATIONALLICENCE 773 0- Cellular and Molecular Life Sciences Springer International Publishing 72/23(2015-12-01), 4633-4651 1420-682X 72:23<4633 2015 72 18