Fluorescence Quenching Assay of Sphingolipid/Phospholipid Phase Separation in Model Membranes

London, Erwin; Brown, Deborah A.; Xu, Xiaolian

doi:10.1016/s0076-6879(00)12915-5

Cited by 18 publications

(19 citation statements)

References 19 publications

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“…T mix is a measure of the stability of domain formation (20,28). We find that T mix defined this way is easier to determine accurately than when defined as the temperature at which domains disappear completely (28). Domain formation can also be assessed by the value of ⌬F/F o at a fixed temperature (Fig.…”

Section: Figmentioning

confidence: 92%

“…In this method the quenching of the fluorescence of membrane-inserted DPH is measured in model membrane vesicles that have the potential to form co-existing domains with different lipid compositions. The vesicles were composed of lipid mixtures containing both lipids having relatively saturated acyl chains (DPPC, SM, or CBs) and 12SLPC, a fluorescence-quenching phospholipid that has phase behavior similar to that of a lipid with unsaturated acyl chains (17,19,20,27,28). The fractional quenching in such mixtures is given by DPH fluorescence intensity (F) normalized to that (F o ) in a mixture in which the quencher lipid is replaced by an unlabeled lipid with unsaturated acyl chains (DOPC).…”

Section: Principles Of the Fluorescence Quenching Assay Of Domainmentioning

confidence: 99%

“…When domain formation occurs in lipid mixtures containing quencher lipid it results in less quenching of DPH than that in lipid mixtures containing quencher lipid but lacking domains. The difference in the extent of quenching arises because saturated lipid-rich domains form quencher-depleted "refuges" within which DPH molecules can fluoresce strongly (28). The degree of domain formation is related to ⌬F/F o , the difference between the fraction of DPH fluorescence that is unquenched in a sample containing saturated acyl chain lipid-rich domains and the fraction of unquenched fluorescence in a sample having the same amount of quencher lipid but lacking such domains (⌬F/F o ϭ F/F o saturated lipid/12SLPC mixture Ϫ F/F o DOPC/12SLPC mixture ) (20).…”

Section: Principles Of the Fluorescence Quenching Assay Of Domainmentioning

confidence: 99%

“…In such experiments a thermal transition can be detected. Above this transition domain formation is abolished (20,28). A transition temperature, T mix , can be defined as the temperature at which the domain mixing/ melting is half-complete (Table I).…”

Section: Figmentioning

confidence: 99%

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Effect of the Structure of Natural Sterols and Sphingolipids on the Formation of Ordered Sphingolipid/Sterol Domains (Rafts)

Xu¹,

Bittman²,

Duportail³

et al. 2001

Journal of Biological Chemistry

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493

211

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Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes. We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E. (2000) Biochemistry 39, 844 -849). In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods. Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains. Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol. Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component. Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains. Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation. The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed.There is strong evidence supporting a model of eukaryotic plasma membrane structure in which domains (rafts) rich in cholesterol and lipids with relatively saturated acyl chains (i.e. sphingolipids) co-exist with domains rich in phospholipids attached to unsaturated acyl chains (1-6). Rafts have a distinct protein composition that is especially enriched in proteins anchored to membranes by saturated acyl chains while relatively depleted of most transmembrane proteins. Rafts have been implicated in numerous cellular processes, including signal transduction, protein and lipid sorting, cellular entry by toxins and viruses, and viral budding (4, 6 -15). Thus, an understanding of the principles that underlie raft formation is important with regard to the study of eukaryotic membrane function.We have shown that sphingolipid/cholesterol domains are likely to exist in the tightly packed liquid-ordered (L o ) state, whereas unsaturated phospholipid-rich domains are more likely to be in the less ordered liquid crystalline (L ␣ ) phase even if they contain large amounts of cholesterol (16 -18). The tight packing of lipids in the L o state gives rafts their characteristic resistance to solubilization by Triton X-100 (1, 18).Cholesterol can promote separation of lipid mixtures into co-existing L ␣ and L o domains (17,19) and is critical for raft formation in cells (2-4, 6). We recently found that the ability of cholesterol to pack tightly with saturated lipids is a key to its abilit...

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Section: Figmentioning

confidence: 92%

Section: Principles Of the Fluorescence Quenching Assay Of Domainmentioning

confidence: 99%

Section: Principles Of the Fluorescence Quenching Assay Of Domainmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

See 2 more Smart Citations

Effect of the Structure of Natural Sterols and Sphingolipids on the Formation of Ordered Sphingolipid/Sterol Domains (Rafts)

Xu¹,

Bittman²,

Duportail³

et al. 2001

Journal of Biological Chemistry

Self Cite

493

211

View full text Add to dashboard Cite

show abstract

“…Our understanding of lipid structure and the formation of specific lipid domains within membranes, however, is almost exclusively based on model membrane systems (5). Although phase separation of domains of liquid-ordered structure is predicted to exist in cellular membranes (6,7), direct demonstration using methodologies such as fluorescence quenching has been difficult to apply to living cells (8). The evidence for the existence of lipid rafts in living cells is largely based on measurements of the clustering (9,10) or diffusion (11,12) of lipid raft proteins, which are secondary to the lipid organization.…”

mentioning

confidence: 99%

Visualizing lipid structure and raft domains in living cells with two-photon microscopy

Gaus

Gratton

Kable

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

493

437

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The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell-cell contacts and cover 10 -15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 m 2 ), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis. membrane domains ͉ macrophages T he lipid raft hypothesis proposes that the lateral organization of cellular membranes is based on the presence of distinct, cholesterol-rich, rigid domains (rafts) (1), which are involved in signal transduction (2), protein sorting, and membrane transport (3, 4). Our understanding of lipid structure and the formation of specific lipid domains within membranes, however, is almost exclusively based on model membrane systems (5). Although phase separation of domains of liquid-ordered structure is predicted to exist in cellular membranes (6, 7), direct demonstration using methodologies such as fluorescence quenching has been difficult to apply to living cells (8). The evidence for the existence of lipid rafts in living cells is largely based on measurements of the clustering (9, 10) or diffusion (11, 12) of lipid raft proteins, which are secondary to the lipid organization.In the present study, we labeled living cells with the fluorescent probe 6-acyl-2-dimethylaminonapthalene (Laurdan), which has been previously used to characterize domain formation and phase separation in model membranes using phospholipid mixtures (13-15) or lipid extracted from cellular membranes (16)(17)(18). Laurdan is an environmentally sensitive fluorescence probe that exhibits a 50-nm red shift as membranes undergo phase transition from gel to fluid, due to altered water penetration into the lipid bilayer (19). Its dipole is aligned parallel to the hydrophobic lipid chains in membranes and is located in both bilayers (20). The probe's fluorescence in water is negligible, and it is not influenced spectroscopically by surface modifications such as lipoprotein binding (20,21). The environmentally induced red shift allows the translation of intensity measurements at different wavelengths into lipid packing orders within the membranes of intact and living cells (20,22,23). Generalized polarization (GP), with a correcting factor G for the experimental setup, is defined analogously to fluorescence polarization by measuring the in...

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Fluorescence Spectroscopy

Sinkeldam¹,

Wilhelmsson²,

Tor³

2016

Fluorescent Analogs of Biomolecular Building Blocks

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Fluorescence Quenching Assay of Sphingolipid/Phospholipid Phase Separation in Model Membranes

Cited by 18 publications

References 19 publications

Effect of the Structure of Natural Sterols and Sphingolipids on the Formation of Ordered Sphingolipid/Sterol Domains (Rafts)

Effect of the Structure of Natural Sterols and Sphingolipids on the Formation of Ordered Sphingolipid/Sterol Domains (Rafts)

Visualizing lipid structure and raft domains in living cells with two-photon microscopy

Fluorescence Spectroscopy

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