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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 living erythrocytes, BODIPY-SLs, but not BODIPY-PC, exhibit differential spectral shift at high concentration.Left, confocal imaging. Freshly isolated erythrocytes were labelled as at Fig. 1, using BODIPY-PC (a), -SM (b-d) or -GlcCer (e-g) at 1 µM (b,e), 2 µM (c,f) or 3 µM (a,d,g), washed and immediately examined by confocal microscopy at 20°C (a-d) or 37°C (e-g). Images were all generated with λexc 488 nm, with simultaneous recording in the green (left; λem 520 nm) and red channels (middle; λem 605 nm), then merged (right). Note that yellow signal in merged images, indicative of ordered clustering (excimers), is essentially absent at 3 µM for BODIPY-PC, weak for -SM and strong for -GlcCer. All scale bars, 2 µm. Right, quantitation of conventional and excimer emission. Intensity profiles were recorded along the paths indicated by the continuous orange lines at left; due to different settings, the minimal baseline values cannot be compared with other figures. Numbers #1-4 refer to the indicated patches. Average red/green emission ratio for BODIPY-SM is <20% at 3 µM (d′), but already >30% for BODIPY-GlcCer at 2 µM (f′).
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pone-0017021-g002: In living erythrocytes, BODIPY-SLs, but not BODIPY-PC, exhibit differential spectral shift at high concentration.Left, confocal imaging. Freshly isolated erythrocytes were labelled as at Fig. 1, using BODIPY-PC (a), -SM (b-d) or -GlcCer (e-g) at 1 µM (b,e), 2 µM (c,f) or 3 µM (a,d,g), washed and immediately examined by confocal microscopy at 20°C (a-d) or 37°C (e-g). Images were all generated with λexc 488 nm, with simultaneous recording in the green (left; λem 520 nm) and red channels (middle; λem 605 nm), then merged (right). Note that yellow signal in merged images, indicative of ordered clustering (excimers), is essentially absent at 3 µM for BODIPY-PC, weak for -SM and strong for -GlcCer. All scale bars, 2 µm. Right, quantitation of conventional and excimer emission. Intensity profiles were recorded along the paths indicated by the continuous orange lines at left; due to different settings, the minimal baseline values cannot be compared with other figures. Numbers #1-4 refer to the indicated patches. Average red/green emission ratio for BODIPY-SM is <20% at 3 µM (d′), but already >30% for BODIPY-GlcCer at 2 µM (f′).

Mentions: The organization of BODIPY-PC, -SM and -GlcCer patches can be probed for clustering-dependent change in spectral properties, known as excimer formation. As recently reported for BODIPY-D-e-LacCer [3] and -SM [31], this phenomenon results in a partial shift of the primary emission peak at λem 520 nm (“green”) to a secondary emission peak at λem 605 nm (“red”), whose combination generates a yellow signal (shown at merge). We therefore looked at green and red fluorescence emission from BODIPY-PC, -SM and -GlcCer patches on erythrocytes, after insertion from 1 µM (usual concentration) to 3 µM, at the optimal temperature for patches formation (20°C for BODIPY-PC and -SM vs 37°C for BODIPY-GlcCer). As shown by Fig. 2, no excimer phenomenon could be detected at BODIPY-PC patches up to the highest concentration tested (Fig. 2a). For BODIPY-SM, excimer formation was barely detected by line scans at 1 µM (Fig. 2b′; see also Fig. 3f), became more visible by direct image inspection at 2 µM (Fig. 2c), and further increased at 3 µM (Fig. 2d,d′), at which concentration red/green emission ratio approached, but did not exceed 20%. For BODIPY-GlcCer, excimer formation was already obvious at 2 µM and yielded emission ratios exceeding 30% (Fig. 2f-g′).


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 living erythrocytes, BODIPY-SLs, but not BODIPY-PC, exhibit differential spectral shift at high concentration.Left, confocal imaging. Freshly isolated erythrocytes were labelled as at Fig. 1, using BODIPY-PC (a), -SM (b-d) or -GlcCer (e-g) at 1 µM (b,e), 2 µM (c,f) or 3 µM (a,d,g), washed and immediately examined by confocal microscopy at 20°C (a-d) or 37°C (e-g). Images were all generated with λexc 488 nm, with simultaneous recording in the green (left; λem 520 nm) and red channels (middle; λem 605 nm), then merged (right). Note that yellow signal in merged images, indicative of ordered clustering (excimers), is essentially absent at 3 µM for BODIPY-PC, weak for -SM and strong for -GlcCer. All scale bars, 2 µm. Right, quantitation of conventional and excimer emission. Intensity profiles were recorded along the paths indicated by the continuous orange lines at left; due to different settings, the minimal baseline values cannot be compared with other figures. Numbers #1-4 refer to the indicated patches. Average red/green emission ratio for BODIPY-SM is <20% at 3 µM (d′), but already >30% for BODIPY-GlcCer at 2 µM (f′).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046177&req=5

pone-0017021-g002: In living erythrocytes, BODIPY-SLs, but not BODIPY-PC, exhibit differential spectral shift at high concentration.Left, confocal imaging. Freshly isolated erythrocytes were labelled as at Fig. 1, using BODIPY-PC (a), -SM (b-d) or -GlcCer (e-g) at 1 µM (b,e), 2 µM (c,f) or 3 µM (a,d,g), washed and immediately examined by confocal microscopy at 20°C (a-d) or 37°C (e-g). Images were all generated with λexc 488 nm, with simultaneous recording in the green (left; λem 520 nm) and red channels (middle; λem 605 nm), then merged (right). Note that yellow signal in merged images, indicative of ordered clustering (excimers), is essentially absent at 3 µM for BODIPY-PC, weak for -SM and strong for -GlcCer. All scale bars, 2 µm. Right, quantitation of conventional and excimer emission. Intensity profiles were recorded along the paths indicated by the continuous orange lines at left; due to different settings, the minimal baseline values cannot be compared with other figures. Numbers #1-4 refer to the indicated patches. Average red/green emission ratio for BODIPY-SM is <20% at 3 µM (d′), but already >30% for BODIPY-GlcCer at 2 µM (f′).
Mentions: The organization of BODIPY-PC, -SM and -GlcCer patches can be probed for clustering-dependent change in spectral properties, known as excimer formation. As recently reported for BODIPY-D-e-LacCer [3] and -SM [31], this phenomenon results in a partial shift of the primary emission peak at λem 520 nm (“green”) to a secondary emission peak at λem 605 nm (“red”), whose combination generates a yellow signal (shown at merge). We therefore looked at green and red fluorescence emission from BODIPY-PC, -SM and -GlcCer patches on erythrocytes, after insertion from 1 µM (usual concentration) to 3 µM, at the optimal temperature for patches formation (20°C for BODIPY-PC and -SM vs 37°C for BODIPY-GlcCer). As shown by Fig. 2, no excimer phenomenon could be detected at BODIPY-PC patches up to the highest concentration tested (Fig. 2a). For BODIPY-SM, excimer formation was barely detected by line scans at 1 µM (Fig. 2b′; see also Fig. 3f), became more visible by direct image inspection at 2 µM (Fig. 2c), and further increased at 3 µM (Fig. 2d,d′), at which concentration red/green emission ratio approached, but did not exceed 20%. For BODIPY-GlcCer, excimer formation was already obvious at 2 µM and yielded emission ratios exceeding 30% (Fig. 2f-g′).

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