Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane[S]

Autor: Mélanie Carquin, Hélène Pollet, Maria Veiga-da-Cunha, Antoine Cominelli, Patrick Van Der Smissen, Francisca N'kuli, Hervé Emonard, Patrick Henriet, Hideaki Mizuno, Pierre J. Courtoy, Donatienne Tyteca
Jazyk: angličtina
Rok vydání: 2014
Předmět:
Zdroj: Journal of Lipid Research, Vol 55, Iss 7, Pp 1331-1342 (2014)
Druh dokumentu: article
ISSN: 0022-2275
DOI: 10.1194/jlr.M048538
Popis: 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 125I-lysenin* binding to erythrocytes upon SM depletion by SMase. The 125I-lysenin* binding isotherm indicated saturation at 3.5 × 106 molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled sub­micrometric 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.
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