Detection of Lipid Domains in Model and Cell Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

Stöckl, Martin; Plazzo, Anna Pia; Korte, Thomas; Herrmann, Andreas

doi:10.1074/jbc.m801418200

Cited by 76 publications

(71 citation statements)

References 59 publications

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“…Decays could be fitted with two lifetimes of about 2.2 ns (s 2 ) and 6.2 ns (s 3 ) with fractional contributions of 0.12 and 0.88, respectively (Table 3), in best agreement with recently reported lifetimes of about 2.5 ns and 6 ns for C6-NBD-PC and for C6-NBD-PS in 1/1 DOPC/DOPS giant vesicles, where the longer lifetime is sensitive to physical properties of the membrane such as lipid packing. 40 We obtained the same results for cuvette experiments using time-resolved fluorescence spectroscopy (data not shown). Therefore, independent of the molecule to which the label is attached (PC or PS), NBD experiences a similar environment in these membranes.…”

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confidence: 71%

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Characterization of lipid bilayers adsorbed on spherical LbL-support

et al. 2009

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Structural and dynamic properties of membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) on layer-by-layer (LbL) polyelectrolyte coated particles were investigated using solid-state nuclear magnetic resonance (NMR) and fluorescence methods. These spherically supported membranes showed structural, dynamic, and elastic properties similar to free-standing membranes as proved by 31 P and 2 H NMR. Small differences between behaviour of PC and PS on LbL support due to interaction with the polyelectrolyte were observed. Fluorescence lifetime imaging microscopy (FLIM) using 7-nitro-2-1,3-benzoxadiazol (NBD) labeled PC and PS showed a stronger impact of the outermost polyelectrolyte (PAH) on the fluorescence lifetimes of NBD-PS compared to NBD-PC. Although small defects in nm range allowing passage of Mn 2+ to both layers of the membrane coat were present, a rather homogeneous coating observed by fluorescence microscopy, complete fluorescence recovery after photobleaching, and NMR results reveal that somewhat continuous lipid bilayers were formed around the LbL particles.

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confidence: 71%

“…40 Therefore, the influence of PAH and LbL surface on C12-NBD-PC and C12-NBD-PS was compared using fluorescence lifetime image microscopy (FLIM). First we measured the fluorescence decay curves for C12-NBD-PC or C12-NBD-PS in POPC/POPS MLVs, i.e.…”

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confidence: 99%

Characterization of lipid bilayers adsorbed on spherical LbL-support

et al. 2009

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“…Chol interacts with the high melting temperature (T m ) sphingolipids (SL) in the membrane, leading to the formation of SL/Chol-enriched microdomains (so-called lipid rafts). These domains are in a more ordered state (usually referred to as liquid-ordered (l o ) phase) than the bulk membrane (liquid-disordered phase (l d )) (3,4). Ceramide (Cer) is an SL formed in stress situations either from sphingomyelin (SM) in rafts or synthesized de novo by serine palmitoyltransferase and ceramide synthase.…”

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confidence: 99%

Cholesterol-rich Fluid Membranes Solubilize Ceramide Domains

Castro¹,

Silva²,

Fedorov³

et al. 2009

Journal of Biological Chemistry

130

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A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.Cholesterol (Chol) 3 is the most abundant sterol in mammalian plasma membrane and has unique biophysical properties (1, 2). Chol interacts with the high melting temperature (T m ) sphingolipids (SL) in the membrane, leading to the formation of SL/Chol-enriched microdomains (so-called lipid rafts). These domains are in a more ordered state (usually referred to as liquid-ordered (l o ) phase) than the bulk membrane (liquid-disordered phase (l d )) (3, 4). Ceramide (Cer) is an SL formed in stress situations either from sphingomyelin (SM) in rafts or synthesized de novo by serine palmitoyltransferase and ceramide synthase. Both of these processes can be induced by diverse stimuli (5). Cer-induced membrane alterations (e.g. raft fusion into large signaling platforms (6)) were proposed to be the mechanism by which this lipid mediates diverse cellular processes, namely apoptosis (7-10). Cer presents an unusually small polar headgroup and in general very high gel-fluid T m (e.g. for palmitoyl-Cer (PCer) it is ϳ90°C) (11)....

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“…Indeed, in some of the above mentioned studies, nanoscale domains were reported (32,48). It has also been suggested that the small size of rafts is due to the coupling of the membrane to the actin cytoskeleton; in the absence of cytoskeleton, as in the case of giant plasma membrane vesicles, large-scale phase separation is observed (46,47,50).…”

Section: A Introductionmentioning

confidence: 95%