Time filter

Source Type

Carquin M.,De Duve Institute And Universitecatholique Of Leuven | Pollet H.,De Duve Institute And Universitecatholique Of Leuven | Veiga-Da-Cunha M.,Matrice Extracellulaire et Dynamique Cellulaire Unit MEDyC | Cominelli A.,De Duve Institute And Universitecatholique Of Leuven | And 7 more authors.
Journal of Lipid Research | Year: 2014

We recently reported that trace insertion of exogenous fl uorescent (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-specifi c nontoxic (NT) fragment of the earthworm toxin, lysenin, fused to the red monomeric fl uorescent protein, mCherry [construct named His-mCherry- NT-lysenin (lysenin)]. Specifi city of lysenin binding was verifi ed with composition-defi ned 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 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 3Dgel. 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. -Carquin, M., H. Pollet, M. Veigada- Cunha, A. Cominelli, P. Van Der Smissen, F. N'kuli, H. Emonard, P. Henriet, H. Mizuno, P. J. Courtoy, and D. Tyteca. Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane. J. Lipid Res. 2014. 55: 1331 - 1342.© 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Dauria L.,De Duve Institute And Universitecatholique Of Leuven | Van Der Smissen P.,De Duve Institute And Universitecatholique Of Leuven | Bruyneel F.,Catholic University of Leuven | Courtoy P.J.,De Duve Institute And Universitecatholique Of Leuven | Tyteca D.,De Duve Institute And Universitecatholique Of Leuven
PLoS ONE | Year: 2011

Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings: We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ~70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance: We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter. © 2011 D′auria et al.

Loading De Duve Institute And Universitecatholique Of Leuven collaborators
Loading De Duve Institute And Universitecatholique Of Leuven collaborators