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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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Differential effect of temperature on boundaries of BODIPY-PC and -GSL micrometric domains in CHO cells.CHO cells were surface-labelled at 4°C with 1 µM BODIPY-PC (a-c) or -D-e-LacCer (d-f), washed and transferred to the indicated temperatures, at which the bottom cell surface was immediately imaged. At left (a-f), confocal imaging. Notice convoluted labelling for BODIPY-PC at 30°C and 37°C, with notches indicated by red arrowheads. All scale bars, 2 µm. At right (a′-f′), quantitation of relative concentrations by line intensity profiles (orange lines at left). Individual, well-defined peaks above fluorescence “baseline” (orange dotted lines at the level of 50 a.u. at right) are numbered from #1 up to 7; clustered patches are indicated by straight brackets; foci below baseline are numbered from #1′ to 2′, and indicated by rounded brackets. Notice that BODIPY-PC concentrates up to 30°C into sharp peaks which vanish at 37°C, whereas BODIPY-D-e-LacCer sharp peaks are best defined at 37°C.
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pone-0017021-g005: Differential effect of temperature on boundaries of BODIPY-PC and -GSL micrometric domains in CHO cells.CHO cells were surface-labelled at 4°C with 1 µM BODIPY-PC (a-c) or -D-e-LacCer (d-f), washed and transferred to the indicated temperatures, at which the bottom cell surface was immediately imaged. At left (a-f), confocal imaging. Notice convoluted labelling for BODIPY-PC at 30°C and 37°C, with notches indicated by red arrowheads. All scale bars, 2 µm. At right (a′-f′), quantitation of relative concentrations by line intensity profiles (orange lines at left). Individual, well-defined peaks above fluorescence “baseline” (orange dotted lines at the level of 50 a.u. at right) are numbered from #1 up to 7; clustered patches are indicated by straight brackets; foci below baseline are numbered from #1′ to 2′, and indicated by rounded brackets. Notice that BODIPY-PC concentrates up to 30°C into sharp peaks which vanish at 37°C, whereas BODIPY-D-e-LacCer sharp peaks are best defined at 37°C.

Mentions: We first reasoned that segregation of domains should somehow be reflected at their boundaries. This was examined after a very brief warming up, from 10°C to 37°C, to minimize internalization (typically within 2 min). BODIPY-PC formed in CHO cells sharply-defined micrometric domains from 10°C to 30°C (Fig. 5a-b′), which showed a comparable, up to ∼5-fold enrichment over the “rest” of the membrane, as in erythrocytes (orange dotted line in intensity profiles at a′,b′), and areas of local depletion could also be clearly seen (arrows at left; continuous pixels below baseline under rounded brackets at right). In contrast, at 37°C, BODIPY-PC boundaries became fuzzy and domains were both less concentrated and much more elongated and indented (Fig. 5c,c′). The opposite temperature effect was observed for the Lo-BODIPY-D-e-LacCer, showing large domains with fuzzy boundaries at 10°C (Fig. 5d,d′) and most obvious peaks of concentration contrasting with foci of local exclusion at 30°C and 37°C (Fig. 5e-f′). As shown at Fig. S4a, BODIPY-L-t-LacCer also concentrated at 10°C into sharply-defined micrometric domains, like BODIPY-PC at this temperature. Incidentally, mammalian cells are not all (or always) operating at 37°C in the body, even without fever or severe hypothermia: for example ∼30°C is a physiological temperature for human erythrocytes when circulating across capillaries of nose mucosa or peripheral skin, as everybody may have experienced.


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)

Differential effect of temperature on boundaries of BODIPY-PC and -GSL micrometric domains in CHO cells.CHO cells were surface-labelled at 4°C with 1 µM BODIPY-PC (a-c) or -D-e-LacCer (d-f), washed and transferred to the indicated temperatures, at which the bottom cell surface was immediately imaged. At left (a-f), confocal imaging. Notice convoluted labelling for BODIPY-PC at 30°C and 37°C, with notches indicated by red arrowheads. All scale bars, 2 µm. At right (a′-f′), quantitation of relative concentrations by line intensity profiles (orange lines at left). Individual, well-defined peaks above fluorescence “baseline” (orange dotted lines at the level of 50 a.u. at right) are numbered from #1 up to 7; clustered patches are indicated by straight brackets; foci below baseline are numbered from #1′ to 2′, and indicated by rounded brackets. Notice that BODIPY-PC concentrates up to 30°C into sharp peaks which vanish at 37°C, whereas BODIPY-D-e-LacCer sharp peaks are best defined at 37°C.
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Related In: Results  -  Collection

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pone-0017021-g005: Differential effect of temperature on boundaries of BODIPY-PC and -GSL micrometric domains in CHO cells.CHO cells were surface-labelled at 4°C with 1 µM BODIPY-PC (a-c) or -D-e-LacCer (d-f), washed and transferred to the indicated temperatures, at which the bottom cell surface was immediately imaged. At left (a-f), confocal imaging. Notice convoluted labelling for BODIPY-PC at 30°C and 37°C, with notches indicated by red arrowheads. All scale bars, 2 µm. At right (a′-f′), quantitation of relative concentrations by line intensity profiles (orange lines at left). Individual, well-defined peaks above fluorescence “baseline” (orange dotted lines at the level of 50 a.u. at right) are numbered from #1 up to 7; clustered patches are indicated by straight brackets; foci below baseline are numbered from #1′ to 2′, and indicated by rounded brackets. Notice that BODIPY-PC concentrates up to 30°C into sharp peaks which vanish at 37°C, whereas BODIPY-D-e-LacCer sharp peaks are best defined at 37°C.
Mentions: We first reasoned that segregation of domains should somehow be reflected at their boundaries. This was examined after a very brief warming up, from 10°C to 37°C, to minimize internalization (typically within 2 min). BODIPY-PC formed in CHO cells sharply-defined micrometric domains from 10°C to 30°C (Fig. 5a-b′), which showed a comparable, up to ∼5-fold enrichment over the “rest” of the membrane, as in erythrocytes (orange dotted line in intensity profiles at a′,b′), and areas of local depletion could also be clearly seen (arrows at left; continuous pixels below baseline under rounded brackets at right). In contrast, at 37°C, BODIPY-PC boundaries became fuzzy and domains were both less concentrated and much more elongated and indented (Fig. 5c,c′). The opposite temperature effect was observed for the Lo-BODIPY-D-e-LacCer, showing large domains with fuzzy boundaries at 10°C (Fig. 5d,d′) and most obvious peaks of concentration contrasting with foci of local exclusion at 30°C and 37°C (Fig. 5e-f′). As shown at Fig. S4a, BODIPY-L-t-LacCer also concentrated at 10°C into sharply-defined micrometric domains, like BODIPY-PC at this temperature. Incidentally, mammalian cells are not all (or always) operating at 37°C in the body, even without fever or severe hypothermia: for example ∼30°C is a physiological temperature for human erythrocytes when circulating across capillaries of nose mucosa or peripheral skin, as everybody may have experienced.

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