Cholesterol-induced fluid-phase immiscibility in membranes.

Sankaram, M. B.; Thompson, T. E.

doi:10.1073/pnas.88.19.8686

Cited by 297 publications

(271 citation statements)

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“…Available data from many experimental studies of the plasma membrane in general suggest cholesterol is the driving force for microdomain formation and cholesterol is not uniformly or homogeneously distributed but that the plasma membrane of living cells consists of areas of cholesterol segregation (regions that are cholesterol-rich and cholesterol-poor) (56,214). Biochemical studies also support this concept and demonstrate that purified cholesterol-rich microdomains isolated from cultured cell (L-cell, MDCK, primary hepatocytes) represent nearly one-third of the plasma membrane, are rich in cholesterol as well as saturated/ monounsaturated fatty acylated phospholipids, and are comprised of physically distinct, liquidordered membrane phases intermediate between fluid liquid-crystalline and rigid gel phases (26,82,102,136,172,182,(215)(216)(217).…”

Section: Summary and Discussionmentioning

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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Section: Summary and Discussionmentioning

confidence: 99%

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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“…Detailed phase diagrams of cholesterol in membranes have been constructed by various groups (Lentz et al, 1980;Sankaram & Thompson, 1991;Huang et al, 1993). In the DPPC/cholesterol system, cholesterol has been proposed to be freely distributed in the bilayer at very low concentrations (<5 mol %).…”

Section: Discussionmentioning

confidence: 99%

Membrane Organization at Low Cholesterol Concentrations: A Study Using 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Cholesterol

1996

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Cholesterol is most often found distributed nonrandomly in the plane of the bilayer, giving rise to cholesterol-rich and -poor domains. Many of these domains are thought to be crucial for the maintenance of membrane structure and function. However, such well-characterized domains generally occur in the membranes that contain relatively large amounts of cholesterol. Cholesterol organization in membranes containing very low amounts of cholesterol has not been investigated extensively. Recent evidence from differential-scanning calorimetric studies suggests that cholesterol may not form uniform monodisperse solutions, as assumed earlier, in the membranes even at very low concentrations. Fluorescent cholesterol analogues, when chosen carefully, offer a powerful approach for studying the distribution and organization of cholesterol in membranes at low concentrations. In this paper, we have studied the organization of cholesterol in membranes at very low concentrations (up to 5 mol % of the total lipid) using a fluorescent cholesterol analogue (NBD-cholesterol) which is labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group at the flexible acyl chain, without any alteration in the structural features necessary for proper membrane incorporation. Our results show that NBD-cholesterol exhibits local organization even at very low concentrations. This is consistent with the recently suggested model of cholesterol organization in membranes at low concentrations, involving the formation of transbilayer, tail-to-tail dimers The fluid mosaic model for biological membranes defines the membrane as an oriented, two-dimensional, viscous solution of proteins and lipids in instantaneous thermodynamic equilibrium (Singer & Nicolson, 1972). Although the basic tenets of this model have stood the test of time, one of its implicit assumptions, that the lipid molecules merely provide the milieu for the membrane proteins and that they are distributed uniformly throughout the bilayer, has been found to be not quite true. In fact, current evidence points out that both transverse and lateral regionalization, which can be described in terms of micro-and macrodomains, are common features of many biological membranes (Curtain et al., 1988;Edidin, 1992;Tocanne, 1992;Glaser, 1993;Jacobson et al., 1995).Cholesterol is one of the membrane components that is found, more often than not, distributed nonrandomly in structural and kinetic domains or pools in both biological and model membranes (Yeagle, 1985;Gennis, 1989;Schroeder et al., 1991;Liscum & Underwood, 1995). The reason for this lies in the rather unusual chemical structure of cholesterol that can by no means be considered a representative component of the "bulk" membrane (see Figure 1). The unusual shape of cholesterol also causes a phase separation in membranes at relatively high concentrations, resulting in the formation of cholesterol-rich and -poor domains that are structurally and compositionally so distinct that they can be observed histochemically and isolated physical...

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“…Such changes might include alterations in the hydrogen-bonding interactions thought to occur in chain-ordered SM (Bruzik, 1988;Sankaram & Thompson, 1991;Bittman et al, 1994). Interestingly, McIntosh et al (1992b) reported that the presence of equimolar cholesterol in SM bilayers alters the hydration pressure relative to that of pure SM bilayers.…”

Section: Cholesterol's Condensation Of Sms and Galcers With Identicalmentioning

confidence: 99%

“…Speyer et al (1989) and Morrow et al (1995)]. In doing so, transient acyl kinks, which enhance the molecular crosssectional area in the hydrocarbon region proximal to the lipid interface, become diminished so that optimal van der Waals interactions can be achieved between the hydrocarbon chain(s) and the α surface of the steroid ring which is held near the interface by a 3-β hydroxyl group (McIntosh et al, 1992a;Vanderkooi, 1994) and may hydrogen bond with the amide group of the sphingolipid (Sankaram & Thompson, 1991;Bittman et al, 1994). Because the cross-sectional area of cholesterol undergoes almost no change in response to increasing surface pressure, the bulk of the condensing effect induced by cholesterol must be due to ordering that occurs in the sphingolipid hydrocarbon region.…”

Section: Cholesterol's Condensation Of Sms and Galcers With Identicalmentioning

confidence: 99%

“…Also, the in-plane lipid-lipid interactions can be isolated from the influences of changing transbilayer compositional distributions that may occur in bilayer systems when cholesterol mole fractions are varied [e.g. Sankaram and Thompson (1991) and Harris et al (1995)]. For these reasons, monolayer investigations of sphingolipid-cholesterol interactions have proven useful (Lund-Katz et al, 1988;Johnston & Chapman, 1988;Grönberg et al, 1991;Slotte et al, 1993;Ali et al, 1994;Bittman et al, 1994).…”

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Cholesterol-Induced Interfacial Area Condensations of Galactosylceramides and Sphingomyelins with Identical Acyl Chains

et al. 1996

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The interfacial interactions occurring between cholesterol and either galactosylceramides (GalCers) or sphingomyelins (SMs) with identical acyl chains have been investigated using Langmuir film balance techniques. Included among the synthesized GalCers and SMs were species containing palmitoyl (16:0), stearoyl (18:0), oleoyl [18:1 ∆9(c) ], nervonoyl [24:1 ∆15(c) ], or linoleoyl [18:2 ∆9,12(c) ] acyl residues. The cholesterol-induced condensations in the average molecular areas of the monolayers were determined by classic mean molecular area vs composition plots as well as by expressing the changes in terms of sphingolipid cross-sectional area reduction over the surface pressure range from 1 to 40 mN/m (at 1 mN/m intervals). The results show that, at surface pressures approximating bilayer conditions (30 mN/m), acyl heterogeneity in naturally occurring SMs (bovine or egg SM) enhanced the area condensation induced by cholesterol compared to their predominant molecular species (e.g. 18:0 SM in bovine SM; 16:0 SM in egg SM). Nonetheless, cholesterol always had a greater condensing effect on SM compared to GalCer when these sphingolipids were acyl chain matched and in similar phase states (prior to mixing with cholesterol). Also, the cholesterol-induced area changes for a given sphingolipid type (e.g. SM or GalCer) were similar whether the acyl chains were saturated, cis-∆9-monounsaturated, or cis-∆9,12-diunsaturated if the sphingolipids were in similar phase states (prior to mixing with cholesterol) and compared at equivalent surface pressures. These results indicate that, under conditions where hydrocarbon structure is matched, the sphingolipid head group plays a dominant role in determining the extent to which cholesterol reduces sphingolipid cross-sectional area. Despite the larger cholesterol-induced area condensations observed in SMs compared to those in GalCers, the molecular-packing densities showed that equimolar GalCercholesterol films were generally packed as tight as or slightly tighter than those of the SMcholesterol films. The results are discussed in terms of a molecular model for sphingolipidcholesterol interactions. Our findings also not only raise questions as to whether cholesterolinduced condensation data provide a reliable measure of the affinity, i.e. interaction strength, between cholesterol and different lipids but also provide insight regarding the stability of sterol/ sphingolipid-rich microdomains thought to exist in caveolae and other cell membrane regions. † This investigation was supported by USPHS Grant GM45928 (R.E.B.) and the Hormel Foundation. The automated Langmuir film balance used in this study receives major support from USPHS Grants HL49180 and HL17371 (H. L. Brockman, P. I.).

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Cholesterol-induced fluid-phase immiscibility in membranes.

Cited by 297 publications

References 23 publications

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Membrane Organization at Low Cholesterol Concentrations: A Study Using 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Cholesterol

Cholesterol-Induced Interfacial Area Condensations of Galactosylceramides and Sphingomyelins with Identical Acyl Chains

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