Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane.
We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous SM, upon binding of a SM-specific nontoxic (NT) fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry [construct named His-mCherry-NT-lysenin (lysenin*)]. Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of (125)I-lysenin* binding to erythrocytes upon SM depletion by SMase. The (125)I-lysenin* binding isotherm indicated saturation at 3.5 × 10(6) molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled sub-micrometric domains on the plasma membrane of partially spread erythrocytes, colocalizing with inserted green BODIPY-SM, and abrogated by SMase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning, and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in RBCs gently suspended in 3D-gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous SM at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo.
CELL Unit, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium.
Mélanie
M
Carquin
CELL Unit, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium.
Hélène
H
Pollet
Laboratory of Physiological Chemistry, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium.
Maria
M
Veiga-da-Cunha
CELL Unit, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium.
Antoine
A
Cominelli
CELL Unit, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium.
Patrick
P
Van Der Smissen
CELL Unit, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium.
Francisca
F
N'kuli
"Matrice Extracellulaire et Dynamique Cellulaire" Unit (MEDyC), CNRS UMR 7369, Université de Reims Champagne-Ardenne, Laboratoire SiRMa, 51 100 Reims, France.
Hervé
H
Emonard
CELL Unit, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium.
Patrick
P
Henriet
Department of Chemistry, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium.
Hideaki
H
Mizuno
CELL Unit, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium.
Pierre J
PJ
Courtoy
CELL Unit, de Duve Institute and Université catholique de Louvain, 1200 Brussels, Belgium.
Donatienne
D
Tyteca
D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism / Q000494:pharmacology*; D014118:Toxins, Biological / Q000494:pharmacology
D013109:Sphingomyelins; D014118:Toxins, Biological; C107040:lysenin
His-mCherry-NT-lysenin; cholesterol; lateral membrane heterogeneity; membrane tension; temperature; toxin; vital confocal imaging