Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types

Parasassi, Tiziana; Loiero, M.; Raimondi, M.; Ravagnan, Giampietro; Gratton, Enrico

doi:10.1016/0005-2736(93)90399-k

Cited by 101 publications

(97 citation statements)

References 38 publications

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“…In lipid vesicles, detergent-resistant membranes in the gel phase can be formed independently of cholesterol, simply by the addition of high amounts of sphingolipid (15). Instead, the lipid composition of the membranes in mammalian cells appears to be such that cholesterol is essential to promote the formation of the liquid-ordered phase, whereas a gel phase does not exist in cell membranes (34). However, it has been observed that mammalian cells can remodel their lipid acyl chains when grown under sterol-limiting conditions.…”

Section: Viruses As Model System To Study Membrane Domains-wementioning

confidence: 99%

Influenza Viruses Select Ordered Lipid Domains during Budding from the Plasma Membrane

Scheiffele¹,

Rietveld²,

Wilk

et al. 1999

Journal of Biological Chemistry

402

386

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Section: Viruses As Model System To Study Membrane Domains-wementioning

confidence: 99%

Influenza Viruses Select Ordered Lipid Domains during Budding from the Plasma Membrane

Scheiffele¹,

Rietveld²,

Wilk

et al. 1999

Journal of Biological Chemistry

402

386

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“…probe affinity for proteins has been shown to be very low, as is its fluorescence intensity in their presence [36,37]. Also, a preferential partitioning of Laurdan in selected phospholipid domains must be excluded [32,39].…”

Section: Absence Of Coexisting Phase Domains In Cell Membranesmentioning

confidence: 99%

“…With the aim of explaining the apparent conflict between the absolute GP value, relatively high and indicative of a rigid environment, and its wavelength dependence, typical of a fluid environment, as measured in cell membranes, we investigated the influence of cholesterol on the phospholipid phases [37,39].…”

Section: The Homogenizing Effect Of Cholesterolmentioning

confidence: 99%

“…Seven cell lines, four primary cell types [36,37], and membranes purified from kidney cortex [38] have been studied with Laurdan. Both Laurdan spectra and GP values in these samples show some peculiarities: (i) the GP values are relatively high, similar to those obtained in phospholipids where the phase state is more than 50% gel (Fig.…”

Section: Absence Of Coexisting Phase Domains In Cell Membranesmentioning

confidence: 99%

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Membrane lipid domains and dynamics as detected by Laurdan fluorescence

1995

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(Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different maxima and shape in the two phospholipid phases, due to differences in the polarity and in the amount of dipolar relaxation, ha bilayers composed of a mixture of gel and liquid-crystalline phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using the generalized polarization (GP). The GP value can be used for the quantitation of each phase. The wavelength dependence of the GP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liquid-crystalline phases was determined. GP measurements performed in several cell lines did not give indications of coexistence of phase domains in their membranes. In natural membranes, Laurdan parameters indicate a homogeneously fluid environment, with restricted molecular motion in comparison with the phospholipid liquid-crystalline phase. The influence of cholesterol on the phase properties of the two phospholipid phases is proposed to be the cause of the phase behavior observed in na~u'al membranes. In bilayers composed of different phospholipids and various cholesterol concentrations, Laurdan response is very similar to that arising from cell membranes. In the absence of cholesterol, from the steady-state and time-resolved measurements of Laurdan in phospholipid vesicles, the condition for the occurrence of separate coexisting domains in the bilayer has been determined: the molecular ratio between the two phases must be in the range between 30% and 70%. Below and above this range, a single homogeneous phase is observed, with the properties of the more concentrated phase, slightly modified by the presence of the other. Moreover, in this concentration range, the calculated dimension of the domains is very small, between 20 and 50 A.

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“…Despite its many applications in model (30,32) and cellular membranes (29,33,34), Laurdan has some disadvantages on a methodological level, including low solubility in aqueous media, low fluorescence intensity, and photo-bleaching, which were overcome by the addition of a carboxyl-headgroup (6-dodecanoyl-2-methylcarboxymethylaminonaphthalene; C-laurdan) (35).…”

mentioning

confidence: 99%

Order of lipid phases in model and plasma membranes

Kaiser

Lingwood

Levental

et al. 2009

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

426

396

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Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165-3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective.generalized polarization value ͉ giant unilamellar vesicle ͉ membrane organization ͉ lipid sorting ͉ lipid raft T he lipid raft hypothesis postulates that selective interactions among sphingolipids, cholesterol, and membrane proteins contribute to lateral membrane heterogeneity (1). A tenet of the model is that small, dynamic cholesterol-sphingolipid-enriched assemblies can be induced to coalesce into larger, more stable structures through clustering of domain components (2). Although experimental data support cholesterol-dependent nano-scale membrane heterogeneity (3-8) and selective domain formation upon raft cross-linking (9-12), the mechanisms that govern such associations in cell membranes remain unclear.On the molecular level, a key feature that is thought to contribute to raft assembly is the propensity of cholesterol to pack tightly with saturated acyl chains of lipids causing them to adopt an extended conformation (13,14). In multi-component model membranes (n Ͼ 2), this interaction can lead to microscopically separate fluid membrane phases: the liquid-ordered (Lo) phase, enriched in saturated (sphingo-)lipids and cholesterol in a highly condensed state, and the liquid-disordered (Ld) phase, enriched in unsaturated glycerophospholipid in a disordered state (15)(16)(17).Several features of the Lo phase in model membranes correspond to the predicted properties of lipid rafts in cel...

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Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types

Cited by 101 publications

References 38 publications

Influenza Viruses Select Ordered Lipid Domains during Budding from the Plasma Membrane

Influenza Viruses Select Ordered Lipid Domains during Budding from the Plasma Membrane

Membrane lipid domains and dynamics as detected by Laurdan fluorescence

Order of lipid phases in model and plasma membranes

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