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Segregation of fluorescent membrane lipids into distinct micrometric domains: evidence for phase compartmentation of natural lipids?

D'auria L, Van der Smissen P, Bruyneel F, Courtoy PJ, Tyteca D - PLoS ONE (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: CELL Unit, de Duve Institute and Université catholique de Louvain, Brussels, Belgium.

ABSTRACT

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.

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BODIPY-PC and -SM enriched micrometric domains show differential sensitivity to endogenous GSL and SM depletion.CHO cells were either kept untreated (a,e; CTL); selectively depleted for GSLs with the GlcCer synthase inhibitor, D-PDMP (b,f) or sphingomyelin with sphingomyelinase (SMase; c,g); or depleted of both using the upstream, dihydroceramide synthase inhibitor, fumonisin B1 (FB1; d,h), then surface-labelled with BODIPY-PC (a-d) or -SM (e-h), washed and immediately examined by confocal microscopy at 10°C. All images are bottom confocal sections recorded at the same laser power and magnification (scale bars, 2 µm). For each panel, intensity profiles along paths indicated by orange lines on confocal images are shown at right (a′-h′), by reference to baseline homogenous labelling (∼50 a.u.; horizontal dotted lines). Notice in control cells similar well-defined patches for the PC and the SM analogs (a,e) with individual sharp peaks (arrowheads #1-4). Most BODIPY-PC micrometric patches/peaks resist GSL depletion by D-PDMP (b) or SM depletion by SMase (c). In contrast, essentially all well-defined micrometric BODIPY-SM patches vanish upon either D-PDMP (f) or SMase (g). For similar properties between BODIPY-PC and -L-t-LacCer, see Fig. S4.
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pone-0017021-g008: BODIPY-PC and -SM enriched micrometric domains show differential sensitivity to endogenous GSL and SM depletion.CHO cells were either kept untreated (a,e; CTL); selectively depleted for GSLs with the GlcCer synthase inhibitor, D-PDMP (b,f) or sphingomyelin with sphingomyelinase (SMase; c,g); or depleted of both using the upstream, dihydroceramide synthase inhibitor, fumonisin B1 (FB1; d,h), then surface-labelled with BODIPY-PC (a-d) or -SM (e-h), washed and immediately examined by confocal microscopy at 10°C. All images are bottom confocal sections recorded at the same laser power and magnification (scale bars, 2 µm). For each panel, intensity profiles along paths indicated by orange lines on confocal images are shown at right (a′-h′), by reference to baseline homogenous labelling (∼50 a.u.; horizontal dotted lines). Notice in control cells similar well-defined patches for the PC and the SM analogs (a,e) with individual sharp peaks (arrowheads #1-4). Most BODIPY-PC micrometric patches/peaks resist GSL depletion by D-PDMP (b) or SM depletion by SMase (c). In contrast, essentially all well-defined micrometric BODIPY-SM patches vanish upon either D-PDMP (f) or SMase (g). For similar properties between BODIPY-PC and -L-t-LacCer, see Fig. S4.

Mentions: As shown by confocal microscopy (Fig. 8b,b′,c,c′; Fig. S4b,c), well-defined patches of BODIPY-PC and -L-t-LacCer could still be observed in either D-PDMP- or SMase-treated CHO cells, although average peak intensity declined and boundaries were more irregular and less sharp (see line intensity profiles at Fig. 8b′c′). The dependence of BODIPY-PC and -L-t-LacCer micrometric assemblies for either endogenous GSLs and SM was confirmed by their disappearance upon combined depletion of GSLs and SM by FB1 treatment (Fig. 8d,d′; Fig. S4d), as if a Ld-phase required only exclusion by a Lo-phase. In contrast, BODIPY-SM domains were strongly impaired by D-PDMP (Fig. 8f,f′), and almost abrogated by SMase (Fig. 8g,g′), suggesting that BODIPY-SM could somehow reflect compartmentation of endogenous SM, and as if BODIPY-SM domains would further depend of a subcompartmentation of the Lo-phase by the more ordered endogenous GSLs.


Segregation of fluorescent membrane lipids into distinct micrometric domains: evidence for phase compartmentation of natural lipids?

D'auria L, Van der Smissen P, Bruyneel F, Courtoy PJ, Tyteca D - PLoS ONE (2011)

BODIPY-PC and -SM enriched micrometric domains show differential sensitivity to endogenous GSL and SM depletion.CHO cells were either kept untreated (a,e; CTL); selectively depleted for GSLs with the GlcCer synthase inhibitor, D-PDMP (b,f) or sphingomyelin with sphingomyelinase (SMase; c,g); or depleted of both using the upstream, dihydroceramide synthase inhibitor, fumonisin B1 (FB1; d,h), then surface-labelled with BODIPY-PC (a-d) or -SM (e-h), washed and immediately examined by confocal microscopy at 10°C. All images are bottom confocal sections recorded at the same laser power and magnification (scale bars, 2 µm). For each panel, intensity profiles along paths indicated by orange lines on confocal images are shown at right (a′-h′), by reference to baseline homogenous labelling (∼50 a.u.; horizontal dotted lines). Notice in control cells similar well-defined patches for the PC and the SM analogs (a,e) with individual sharp peaks (arrowheads #1-4). Most BODIPY-PC micrometric patches/peaks resist GSL depletion by D-PDMP (b) or SM depletion by SMase (c). In contrast, essentially all well-defined micrometric BODIPY-SM patches vanish upon either D-PDMP (f) or SMase (g). For similar properties between BODIPY-PC and -L-t-LacCer, see Fig. S4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046177&req=5

pone-0017021-g008: BODIPY-PC and -SM enriched micrometric domains show differential sensitivity to endogenous GSL and SM depletion.CHO cells were either kept untreated (a,e; CTL); selectively depleted for GSLs with the GlcCer synthase inhibitor, D-PDMP (b,f) or sphingomyelin with sphingomyelinase (SMase; c,g); or depleted of both using the upstream, dihydroceramide synthase inhibitor, fumonisin B1 (FB1; d,h), then surface-labelled with BODIPY-PC (a-d) or -SM (e-h), washed and immediately examined by confocal microscopy at 10°C. All images are bottom confocal sections recorded at the same laser power and magnification (scale bars, 2 µm). For each panel, intensity profiles along paths indicated by orange lines on confocal images are shown at right (a′-h′), by reference to baseline homogenous labelling (∼50 a.u.; horizontal dotted lines). Notice in control cells similar well-defined patches for the PC and the SM analogs (a,e) with individual sharp peaks (arrowheads #1-4). Most BODIPY-PC micrometric patches/peaks resist GSL depletion by D-PDMP (b) or SM depletion by SMase (c). In contrast, essentially all well-defined micrometric BODIPY-SM patches vanish upon either D-PDMP (f) or SMase (g). For similar properties between BODIPY-PC and -L-t-LacCer, see Fig. S4.
Mentions: As shown by confocal microscopy (Fig. 8b,b′,c,c′; Fig. S4b,c), well-defined patches of BODIPY-PC and -L-t-LacCer could still be observed in either D-PDMP- or SMase-treated CHO cells, although average peak intensity declined and boundaries were more irregular and less sharp (see line intensity profiles at Fig. 8b′c′). The dependence of BODIPY-PC and -L-t-LacCer micrometric assemblies for either endogenous GSLs and SM was confirmed by their disappearance upon combined depletion of GSLs and SM by FB1 treatment (Fig. 8d,d′; Fig. S4d), as if a Ld-phase required only exclusion by a Lo-phase. In contrast, BODIPY-SM domains were strongly impaired by D-PDMP (Fig. 8f,f′), and almost abrogated by SMase (Fig. 8g,g′), suggesting that BODIPY-SM could somehow reflect compartmentation of endogenous SM, and as if BODIPY-SM domains would further depend of a subcompartmentation of the Lo-phase by the more ordered endogenous GSLs.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: CELL Unit, de Duve Institute and Université catholique de Louvain, Brussels, Belgium.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus