title: 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.
authors.affiliation: CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Carquin
authors.affiliation: CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium.
authors.forename: Louise
authors.initials: L
authors.lastname: Conrard
authors.affiliation: CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium.
authors.forename: Hélène
authors.initials: H
authors.lastname: Pollet
authors.affiliation: CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium.
authors.forename: Patrick
authors.initials: P
authors.lastname: Van Der Smissen
authors.affiliation: CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium.
authors.forename: Antoine
authors.initials: A
authors.lastname: Cominelli
authors.affiliation: Laboratory of Physiological Chemistry, de Duve Institute and Université Catholique de Louvain, 1200, Brussels, Belgium.
authors.forename: Maria
authors.initials: M
authors.lastname: Veiga-da-Cunha
authors.affiliation: CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium.
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Courtoy
authors.affiliation: CELL Unit, de Duve Institute and Université Catholique de Louvain, UCL B1.75.05, Avenue Hippocrate, 75, 1200, Brussels, Belgium. donatienne.tyteca@uclouvain.be.
authors.forename: Donatienne
authors.initials: D
authors.lastname: Tyteca
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D001896:Boron Compounds / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D032446:Myoblasts / Q000378:metabolism; D013109:Sphingomyelins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001427:Bacterial Toxins; D001896:Boron Compounds; D006460:Hemolysin Proteins; D013109:Sphingomyelins; C020452:Clostridium perfringens theta-toxin; D002784:Cholesterol
keywords: 125I-toxin; BODIPY-sphingomyelin; C2C12 myoblasts; His-mCherry-theta-D4; Lateral membrane heterogeneity; Membrane tension; Temperature; Vital confocal imaging