Fluorescence of .DELTA.5,7,9(11),22-ergostatetraen-3.beta.-ol in micelles, sterol carrier protein complexes, and plasma membranes

Fischer, Reinhard; Cowlen, Matthew S.; Dempsey, Mary E.; Schroeder, Friedhelm

doi:10.1021/bi00334a037

Cited by 74 publications

(65 citation statements)

References 64 publications

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“…The naturally-occurring fluorescent sterol DHE (19,21) has proven a powerful probe for use in investigating membrane structure and cholesterol domain dynamics in vitro (8,54,55,65,70,102,172), for examining cholesterol-protein interactions in vitro (2,39,96,101,102,108,205), and for first time visualization in real-time of the distribution of cholesterol in the plasma membranes in living cells (70,203,204). Successful application of the fluorescent DHE to investigation of the function and organization of sterols in membranes, especially lipid raft/ caveolae microdomains of living cells, requires the use of highly purified DHE.…”

Section: Summary and Discussionmentioning

confidence: 99%

“…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, near 20-30 nM (1)(2)(3)(4)(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)(7)(8)(9).…”

Section: Historical Perspectivementioning

confidence: 99%

“…These studies showed that DHE was localized in the surface monolayer surrounding the neutral lipid cores of triglyceride/cholesteryl ester rich lipoproteins similarly as cholesterol. In addition, DHE and a similar synthetic fluorescent cholesterol analog (cholestatrienol, CTE) have been used to examine the structure of the sterol binding site of cholesterol-binding proteins such as sterol carrier protein-2 (SCP-2) (2,39,102-105) and liver fatty acid binding protein (L-FABP) (2,106,107). SCP-2 and L-FABP generally bound one mole of fluorescent sterol/mole protein.…”

Section: Monomeric Vs Crystalline Dhementioning

confidence: 99%

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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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Section: Summary and Discussionmentioning

confidence: 99%

Section: Historical Perspectivementioning

confidence: 99%

Section: Monomeric Vs Crystalline Dhementioning

confidence: 99%

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Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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“…Fluorescence and light scattering direct binding assays indicate that L-FABP binds cholesterol and a fluorescent sterol (dehydroergosterol) with low affinity (i.e. submicromolar K d levels) (38,44,45). Although L-FABP poorly competes for binding, extracting, or transferring sterol from model membranes (46 -48), it nevertheless enhances transfer of sterol in vitro from purified plasma membrane vesicles, albeit not as strongly as sterol carrier protein-2 (46).…”

Section: Discussionmentioning

confidence: 99%

Decreased Liver Fatty Acid Binding Capacity and Altered Liver Lipid Distribution in Mice Lacking the Liver Fatty Acid-binding Protein Gene

Martin

Danneberg

Kumar

et al. 2003

Journal of Biological Chemistry

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156

183

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Although liver fatty acid-binding protein (L-FABP) isan important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2-to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected family (1-3), is found in the liver, intestine, and kidney, but only in liver is it not co-expressed with other members of its family. L-FABP is known to bind fatty acids and various other hydrophobic molecules, although its actual contribution to the lipid-binding capacity of liver cytosol is not known. Given that L-FABP is expressed at very high levels (2-5% of cytosolic protein) in the differentiated hepatocyte (4, 5) and that these levels correlate well with lipid metabolism (2), it can be speculated that L-FABP contributes considerably to hepatic lipidbinding and lipid metabolism. Work with cell-free systems and transfected cells has further strengthened this view. For example, in cell-free preparations L-FABP was shown to stimulate the esterification of oleic acid while inhibiting that of palmitic acid (6). L cells overexpressing L-FABP show increased rates of fatty acid uptake and esterification (7) as well as increased contents of phospholipid and cholesterol esters (8, 9). HepG2 hepatoma cells expressing an L-FABP antisense RNA showed a dose-dependent reduction of fatty acid uptake (10). Furthermore, overexpression of L-FABP in McA-RH7777 hepatoma cells incubated with palmitic acid decreased the synthesis and secretion of triglycerides while increasing beta oxidation and the secretion of apolipoprotein B100 (11). Thus, the various in vitro systems have allowed researchers to propose specific functions of L-FABP in vivo.However, in vitro studies of FABPs have inherent limitations. The only firmly established function of FABPs is the reversible binding of hydrophobic ligands, and these proteins do not exhibit any enzymatic function or energy requirement. This suggests that these proteins play passive (facilitative) roles that, almost by definition, are strongly dependent on the cellular context. One context of the highly expressed L-FABP is the highly differentiated hepatocyte, a cell type featuring an intense lipid metabolism that is not eas...

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“…These emission characteristics of crystalline DHE were not due to the presence of impurities. The onset of the red-shift was concomitant with the formation of DHE microcrystals in aqueous buffers (27,30) at concentrations similar to that where cholesterol has been shown to form microcrystals in aqueous buffers (49,(61)(62)(63). Several techniques were applied to determine whether the change in spectral characteristics from ethanol to water was due to formation of microcrystals or of micelles.…”

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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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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Fluorescence of .DELTA.5,7,9(11),22-ergostatetraen-3.beta.-ol in micelles, sterol carrier protein complexes, and plasma membranes

Cited by 74 publications

References 64 publications

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Decreased Liver Fatty Acid Binding Capacity and Altered Liver Lipid Distribution in Mice Lacking the Liver Fatty Acid-binding Protein Gene

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

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