Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence

Parasassi, Tiziana; Stefano, M. Di; Loiero, M.; Ravagnan, Giampietro; Gratton, Enrico

doi:10.1016/s0006-3495(94)80763-5

Cited by 222 publications

(199 citation statements)

References 23 publications

(68 reference statements)

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“…In our LUV experiments, we found a strong correlation between membrane composition and order as expected (36). Increasing amounts of partially saturated lipids and cholesterol led to higher membrane packing (Fig.…”

Section: Correlation Of Model Membrane Composition and Ordersupporting

confidence: 86%

Order of lipid phases in model and plasma membranes

Kaiser

Lingwood

Levental

et al. 2009

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

412

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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Section: Correlation Of Model Membrane Composition and Ordersupporting

confidence: 86%

Order of lipid phases in model and plasma membranes

Kaiser

Lingwood

Levental

et al. 2009

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

412

396

View full text Add to dashboard Cite

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“…8) suggested that lyso-PPC caused a decrease in the interaction of water with the bilayer since the laurdan emission spectrum is sensitive to the amount and mobility of water molecules in the region of the phospholipid glycerol backbone (46,47). Importantly, this effect of lyso-PPC was not blocked by cobalt (Fig.…”

Section: Discussionmentioning

confidence: 92%

Mechanisms by Which Elevated Intracellular Calcium Induces S49 Cell Membranes to Become Susceptible to the Action of Secretory Phospholipase A2

Wilson¹,

Waldrip²,

Nielson³

et al. 1999

Journal of Biological Chemistry

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Exposure of S49 lymphoma cells to exogenous group IIA or V secretory phospholipase A 2 (sPLA 2 ) caused an initial release of fatty acid followed by resistance to further hydrolysis by the enzyme. This refractoriness was overcome by exposing cells to palmitoyl lysolecithin. This effect was specific in terms of lysophospholipid structure. Induction of membrane susceptibility by lysolecithin involved an increase in cytosolic calcium and was duplicated by incubating the cells with calcium ionophores such as ionomycin. Lysolecithin also activated cytosolic phospholipase A 2 (cPLA 2 ). Inhibition of this enzyme attenuated the ability of lysolecithin (but not ionomycin) to induce susceptibility to sPLA 2 . Lysolecithin or ionomycin caused concurrent hydrolysis of both phosphatidylethanolamine and phosphatidylcholine implying that transbilayer movement of phosphatidylethanolamine occurred upon exposure to these agents but that susceptibility is not simply due to exposure of a preferred substrate (i.e. phosphatidylethanolamine) to the enzyme. Microvesicles were apparently released from the cells upon addition of lysolecithin or ionomycin. Both these vesicles and the remnant cell membranes were susceptible to sPLA 2 . Together these data suggest that lysolecithin induces susceptibility through both cPLA 2 -dependent and -independent pathways. Whereas elevated cytosolic calcium was required for both pathways, it was sufficient only for the cPLA 2 -independent pathway. This cPLA 2 -independent pathway involved changes in cell membrane structure associated with transbilayer phospholipid migration and microvesicle release.

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“…The LAURDAN probe diffuses into tissue lipids and, due to its virtually complete partitioning into lipids with respect to the aqueous environment, its labeling persists while the tissue is imaged in its buffer. The value of LAURDAN GP allows the identification of LDL particles in the GP image because the cholesterol-rich LDL displays a GP value higher than that of vascular cell membranes [33].…”

Section: Discussionmentioning

confidence: 99%

Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides

Parasassi

Durbin

et al. 2000

Free Radical Biology and Medicine

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Abstract-Oxidatively modified LDL mimics several aspects of atherogenesis. In this disease, degradation of the matrix proteins' network also occurs. By a new morphological ex vivo approach, not requiring sample processing, we explored the relationship between the degradation of matrix protein and oxidatively modified LDL. Two-photon excitation fluorescence microscopy images of fresh cross-section rings of rat aorta, acquired while the sample was maintained in a glucose-and oxygen-supplemented buffer, showed straight, parallel, thick, long extracellular matrix proteins. Traditional microscopic examination, requiring sample fixation and staining, shows smaller and curved fibers. Instead, we observed curved and broken fibers after a 30-min incubation of aorta with either LDL containing lipid hydroperoxides, or tert-butyl-hydroperoxide. The adhesion of LDL to the endothelium and its internalization was directly visualized by using a lipid fluorophore. The damage to aorta matrix proteins induced by LDL and tert-butylhydroperoxide was fully prevented by antioxidants, such as ascorbate or Trolox C, or inhibitors of proteases. The image spectroscopy of the fibers' autofluorescence (polarization and lifetime) revealed an increased mobility of the fluorescent cross-link in fibers. Damaged matrix proteins were also imaged in aorta samples from apolipoprotein E knock-out mice. Our ex vivo images directly visualized the activation of a fast redox-sensitive proteolytic process in the arterial wall triggered by lipid hydroperoxides in LDL.

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Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence

Cited by 222 publications

References 23 publications

Order of lipid phases in model and plasma membranes

Order of lipid phases in model and plasma membranes

Mechanisms by Which Elevated Intracellular Calcium Induces S49 Cell Membranes to Become Susceptible to the Action of Secretory Phospholipase A2

Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides

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