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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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In CHO cells, only BODIPY-D-e-SLs form micrometric patches competent for excimer formation.CHO cells were surface-labelled at low temperature with 5 µM of the indicated BODIPY-lipids, washed and immediately examined by confocal microscopy at 10°C to prevent endocytosis. Bottom confocal sections are shown. Images were recorded in the green channel (left) at the usual intensity, then in the red channel at 30-times higher laser power (middle) and merged (right). Whereas excimer formation (yellow signal at right) is obvious for BODIPY-SM (f) and BODIPY-GSLs with natural stereochemistry [-GlcCer (i) and -D-e-LacCer (l)], no spectral shift is observed using BODIPY-PC (c) and a GSL with artificial stereochemistry, -L-t-LacCer (o). For the two latter derivatives, notice convoluted wavy labelling, with notches indicated by red arrowheads. All scale bars, 2 µm.
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pone-0017021-g004: In CHO cells, only BODIPY-D-e-SLs form micrometric patches competent for excimer formation.CHO cells were surface-labelled at low temperature with 5 µM of the indicated BODIPY-lipids, washed and immediately examined by confocal microscopy at 10°C to prevent endocytosis. Bottom confocal sections are shown. Images were recorded in the green channel (left) at the usual intensity, then in the red channel at 30-times higher laser power (middle) and merged (right). Whereas excimer formation (yellow signal at right) is obvious for BODIPY-SM (f) and BODIPY-GSLs with natural stereochemistry [-GlcCer (i) and -D-e-LacCer (l)], no spectral shift is observed using BODIPY-PC (c) and a GSL with artificial stereochemistry, -L-t-LacCer (o). For the two latter derivatives, notice convoluted wavy labelling, with notches indicated by red arrowheads. All scale bars, 2 µm.

Mentions: Since erythrocytes are very special cells, we extended our investigations to CHO cells, where BODIPY-SM micrometric domains were observed and shown to form excimers [31]. BODIPY-PC and -D-e-LacCer superficial patches were readily detected on CHO cells as well. As shown by resistance to K+-depletion (Fig. S3b,e) and latrunculin B treatment (Fig. S3c,f), these patches genuinely reflected packaging at the PM, and not peripheral endosomes [41] nor actin-dependent surface extensions. To sensitize excimer formation in CHO cells, BODIPY-lipids were inserted at a higher concentration (5 µM). As shown by Fig. 4b,c,n,o, BODIPY-PC and -L-t-LacCer micrometric patches were incompetent to form excimers, in contrast to BODIPY-SM (Fig. 4e,f), and especially -D-e-LacCer (Fig. 4k,l) as well as -GlcCer (Fig. 4h,i). These data indicated that, in both cell types, micrometric domains labelled by lipid analogs differed in intrinsic ordering, with the following ranking: BODIPY-GSLs > -SM > -PC or -L-t-LacCer.


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)

In CHO cells, only BODIPY-D-e-SLs form micrometric patches competent for excimer formation.CHO cells were surface-labelled at low temperature with 5 µM of the indicated BODIPY-lipids, washed and immediately examined by confocal microscopy at 10°C to prevent endocytosis. Bottom confocal sections are shown. Images were recorded in the green channel (left) at the usual intensity, then in the red channel at 30-times higher laser power (middle) and merged (right). Whereas excimer formation (yellow signal at right) is obvious for BODIPY-SM (f) and BODIPY-GSLs with natural stereochemistry [-GlcCer (i) and -D-e-LacCer (l)], no spectral shift is observed using BODIPY-PC (c) and a GSL with artificial stereochemistry, -L-t-LacCer (o). For the two latter derivatives, notice convoluted wavy labelling, with notches indicated by red arrowheads. All scale bars, 2 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046177&req=5

pone-0017021-g004: In CHO cells, only BODIPY-D-e-SLs form micrometric patches competent for excimer formation.CHO cells were surface-labelled at low temperature with 5 µM of the indicated BODIPY-lipids, washed and immediately examined by confocal microscopy at 10°C to prevent endocytosis. Bottom confocal sections are shown. Images were recorded in the green channel (left) at the usual intensity, then in the red channel at 30-times higher laser power (middle) and merged (right). Whereas excimer formation (yellow signal at right) is obvious for BODIPY-SM (f) and BODIPY-GSLs with natural stereochemistry [-GlcCer (i) and -D-e-LacCer (l)], no spectral shift is observed using BODIPY-PC (c) and a GSL with artificial stereochemistry, -L-t-LacCer (o). For the two latter derivatives, notice convoluted wavy labelling, with notches indicated by red arrowheads. All scale bars, 2 µm.
Mentions: Since erythrocytes are very special cells, we extended our investigations to CHO cells, where BODIPY-SM micrometric domains were observed and shown to form excimers [31]. BODIPY-PC and -D-e-LacCer superficial patches were readily detected on CHO cells as well. As shown by resistance to K+-depletion (Fig. S3b,e) and latrunculin B treatment (Fig. S3c,f), these patches genuinely reflected packaging at the PM, and not peripheral endosomes [41] nor actin-dependent surface extensions. To sensitize excimer formation in CHO cells, BODIPY-lipids were inserted at a higher concentration (5 µM). As shown by Fig. 4b,c,n,o, BODIPY-PC and -L-t-LacCer micrometric patches were incompetent to form excimers, in contrast to BODIPY-SM (Fig. 4e,f), and especially -D-e-LacCer (Fig. 4k,l) as well as -GlcCer (Fig. 4h,i). These data indicated that, in both cell types, micrometric domains labelled by lipid analogs differed in intrinsic ordering, with the following ranking: BODIPY-GSLs > -SM > -PC or -L-t-LacCer.

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