A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles

Schroeder, Friedhelm; Barenholz, Yechezkel; Gratton, Enrico; Thompson, T. E.

doi:10.1021/bi00383a007

Cited by 99 publications

(106 citation statements)

References 36 publications

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“…DHE: phospholipid molar ratio of 0.54) ( Table I). It should be noted that in membranes composed of POPC:DHE at 65:35, the DHE undergoes significant self-quenching by Forster energy transfer (29). The fluorescence emission intensity was 63% lower than expected, based on comparison with that of DHE in membranes composed of POPC:DHE:cholesterol of 65:5:30 ( Fig.…”

Section: Synthesis and Purification Of Dhe-to Determine Structuralmentioning

confidence: 75%

“…While at low mol % the sterol is uniformly dispersed/solubilized in the membrane, and increasing the sterol:phospholipid molar ratio from 0.25 to 1 results in formation of interdigitated, transbilayer sterol dimers (66). Over the same concentration, DHE self-quenches in model membranes by Forster energy transfer (29,30). The Forster energy transfer distance for this homotransfer between face-to-face oriented DHE molecules is 13.3 Å (30).…”

Section: Discussionmentioning

confidence: 99%

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Fluorescence and Multiphoton Imaging Resolve Unique Structural Forms of Sterol in Membranes of Living Cells

McIntosh¹,

Gallegos²,

Atshaves³

et al. 2003

Journal of Biological Chemistry

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113

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Although cholesterol is an essential component of mammalian membranes, resolution of cholesterol organization in membranes and organelles (i.e. lysosomes) of living cells is hampered by the paucity of nondestructive, nonperturbing methods providing real time structural information. Advantage was taken of the fact that the emission maxima of a naturally occurring fluorescent sterol (dehydroergosterol) were resolvable into two structural forms, monomeric (356 and 375 nm) and crystalline (403 and 426 nm). Model membranes (sterol:phospholipid ratios in the physiological range, e.g. 0.5-1.0), subcellular membrane fractions (plasma membranes, lysosomal membranes, microsomes, and mitochondrial membranes), and lipid rafts/caveolae (plasma membrane cholesterol-rich microdomain purified by a nondetergent method) contained primarily monomeric sterol and only small quantities (i.e. 1-5%) of the crystalline form. In contrast, the majority of sterol in isolated lysosomes was crystalline. However, addition of sterol carrier protein-2 in vitro significantly reduced the proportion of crystalline dehydroergosterol in the isolated lysosomes. Multiphoton laser scanning microscopy (MPLSM) of living L-cell fibroblasts cultured with dehydroergosterol for the first time provided real time images showing the presence of monomeric sterol in plasma membranes, as well as other intracellular membrane structures of living cells. Furthermore, MPLSM confirmed that crystalline sterol colocalized in highest amounts with LysoTracker Green, a lysosomal marker dye. Although crystalline sterol was also detected in the cytoplasm, the extralysosomal crystalline sterol did not colocalize with BODIPY FL C 5 -ceramide, a Golgi marker, and crystals were not associated with the cell surface membrane. These noninvasive, nonperturbing methods demonstrated for the first time that multiple structural forms of sterol normally occurred within membranes, membrane microdomains (lipid rafts/ caveolae), and intracellular organelles of living cells, both in vitro and visualized in real time by MPLSM.Cholesterol is essential for optimal membrane transport, receptor-effector coupling, cell recognition, and other eukaryotic cellular processes (reviewed in Ref. 1). Increasing evidence indicates that plasma membrane cholesterol is organized into lateral and transbilayer cholesterol-rich microdomains (reviewed in Ref.2) such as lipid rafts and caveolae (reviewed in Refs. 2-6). These domains contain proteins involved in multiple cellular functions including signaling and cholesterol transport. For example, overexpression of caveolin-1 in mice stimulates high density lipoprotein uptake via plasma membrane caveolae and markedly increases plasma high density lipoprotein-cholesterol (8). In contrast, caveolin-1-deficient mice are lean and resistant to diet-induced obesity but show hypertriglyceridemia and exhibit vascular abnormalities (9 -11). There is a strong association of cholesterol abnormalities with cytotoxicity, sickle cell acanthacytosis, Niemann-Pick C disease, Alzhe...

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Section: Synthesis and Purification Of Dhe-to Determine Structuralmentioning

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Fluorescence and Multiphoton Imaging Resolve Unique Structural Forms of Sterol in Membranes of Living Cells

McIntosh¹,

Gallegos²,

Atshaves³

et al. 2003

Journal of Biological Chemistry

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113

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“…Fluorescent sterols, such as dehydroergosterol, are excellent nonperturbing probe molecules for sterols in model [40][41][42] and biological membranes [4][5][6]. Dehydroergosterol offers significant advantages over nitroxide or deuterated sterols [42,43].…”

Section: Resultsmentioning

confidence: 99%

“…Dehydroergosterol offers significant advantages over nitroxide or deuterated sterols [42,43]. Because the determination of transbilayer distribution of dehydroergosterol depends on trinitrophenylation of the exofacial amino groups, it was prudent to assess the effect of fatty acid supplementation on the plasma membrane purity.…”

Section: Resultsmentioning

confidence: 99%

Polyunsaturated fatty acids alter sterol transbilayer domains in LM fibroblast plasma membrane

Sweet

Schroeder

1988

FEBS Letters

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Sterols are asymmetrically distributed between the leaflets of animal cell plasma membranes. Although transbilayer migration of sterols is extremely rapid, s to rain, previous experimental manipulations have not altered their transmembrane steady-state distribution. However, the effect of polyunsaturated fatty acids has not been reported. When cultured in a lipid-free, chemically defined culture medium, LM fibroblasts do not synthesize polyunsaturated fatty acids but will incorporate polyunsaturated fatty acids into their plasma membranes if supplied in the medium. Sterol transbilayer distribution in LM plasma membranes was determined from quenching of fluorescence of dehydroergosterol by trinitrophenyl groups selectively attached to the exofacial leaflet. When cells are cultured in lipid-free media, 28.1% of the plasma membrane sterol is located in the exofacial (outside) leaflet. In contrast, when cells are cultured with linoleate-or linolenatesupplemented medium, 71 ;8% and 75.5% of the plasma membrane sterol is exofacial, respectively.

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“…The usefulness of DHE and cholestatrienol (CTE) as fluorescent cholesterol analogs in model membranes in a series of investigations spanning several decades demonstrated the usefulness of these probes to monitor cholesterol structural (polarization, limiting anisotropy, order) and dynamic (lifetime, rotational rate) properties in membranes (12,65,70,95,96,108,(110)(111)(112)(113)(114)(115)(116)(117)(118)(119)(120). The lifetime studies resolved for the first time at least two DHE domains in model membranes, one less sensitive to the aqueous than the other, and that these domains were dependent on the lipid composition, temperature, and other properties of the membrane (54,119).…”

Section: Model Membranesmentioning

confidence: 99%

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent of dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular sterol trafficking in living organisms. DHE is a naturally-occurring, fluorescent sterol analog that faithfully mimics many of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins, is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock solution to cell culture media is effective, but this process forms an aqueous dispersion of dehydroergosterol crystals which can result in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques can deliver large quantities of monomeric dehydroergosterol with significant distribution into the plasma membrane. The properties and behavior of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging in living cells indicate that this naturally-occurring fluorescent sterol is a useful mimic for probing the properties of cholesterol in these systems. Keywordsdehydroergosterol; cholesterol; ergosterol; fluorescent sterols; microscopy Historical PerspectiveIn order to resolve the dynamics and factors governing cholesterol trafficking within cells, it is important to recognize that the cholesterol molecule has very few polar constituents (Fig. 1A). Consequently, cholesterol is poorly soluble in aqueous environments such as cytosol as evidenced by critical micellar concentrations of cholesterol and other sterols being very low, *To whom correspondence should be addressed: Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 862-1433, FAX: (979) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript near 20-30 nM (1-5). The physiological impact of cholesterol's low aqueous solubility is that spontaneous cholesterol desorption from the cytofacial leaflet of the plasma membrane or lysosomal membrane into the cytosol for transfer to intracellular sites for esterification, oxidation, or secretion is extremely slow (t 1/2 3 hours-days) (6-9). Therefore, it is important to resolve factors...

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A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles

Cited by 99 publications

References 36 publications

Fluorescence and Multiphoton Imaging Resolve Unique Structural Forms of Sterol in Membranes of Living Cells

Fluorescence and Multiphoton Imaging Resolve Unique Structural Forms of Sterol in Membranes of Living Cells

Polyunsaturated fatty acids alter sterol transbilayer domains in LM fibroblast plasma membrane

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

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