Fluorescent Detection of Lipid Droplets and Associated Proteins

Listenberger, Laura L.; Brown, Deborah A.

doi:10.1002/0471143030.cb2402s35

Cited by 176 publications

(170 citation statements)

References 28 publications

(35 reference statements)

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“…Essentially identical results regarding LD occurrence (supplemental Fig. S6G) were obtained when 10 M oleate was supplied in a more physiological way, after complexion with bovine serum albumin at a ratio of 6:1 (48). Taken together, these results show that fatty acid-induced formation of LD is a cPLA 2 ␣-dependent process and suggest that the enzyme may be necessary for roles other than generating fatty acids for TAG and cholesteryl ester synthesis.…”

Section: Resultssupporting

confidence: 73%

Group IVA Phospholipase A2 Is Necessary for the Biogenesis of Lipid Droplets

Gubern

Casas

Barceló-Torns

et al. 2008

Journal of Biological Chemistry

107

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Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A 2 inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A 2 (cPLA 2 ␣). Knocking down cPLA 2 ␣ expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA 2 ␣ fusion protein (EGFP-cPLA 2 ) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA 2 ␣ at Ser 505 . Transfection of a S505A mutant cPLA 2 ␣ showed that phosphorylation at Ser 505 is key for enzyme activity and LD formation. cPLA 2 ␣ contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA 2 ␣ inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA 2 ␣ in the formation of nascent LD from the endoplasmic reticulum.

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Section: Resultssupporting

confidence: 73%

Group IVA Phospholipase A2 Is Necessary for the Biogenesis of Lipid Droplets

Gubern

Casas

Barceló-Torns

et al. 2008

Journal of Biological Chemistry

107

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“…Their spherical shape, and the fact that BODIPY itself is a marker for neutral lipid storage bodies (Listenberger and Brown, 2007) suggests that these organelles are lipid droplets (LD). To test this hypothesis, we co-stained HeLa cells with Nile Red, a prominent marker for neutral lipid stores We found co-localization between some BCh2 and BCh1 with Nile Red, while DHE was absent from most of the structures stained by the droplet marker ( Fig.…”

Section: Steady-state Distribution Of Fluorescent Sterols In Living Cmentioning

confidence: 99%

“…To further assess the potential of BCh2 as a cholesterol analog, we have now undertaken a direct comparison of the membrane distribution and intracellular trafficking of DHE and BCh2 by duallabeling multicolor fluorescence microscopy in control and fatty acid treated cells that contain lipid droplets (LD) (Listenberger and Brown, 2007). In order to rule out that specific photophysical characteristics of the BODIPY-moiety affect the quantification of sterol distribution, we measured a variety of fluorescence properties of BCh2, including its radiative lifetime and photobleaching kinetics on a pixel-by-pixel basis in living cells.…”

Section: Introductionmentioning

confidence: 99%

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

Wüstner

Solanko

Sokol

et al. 2011

Chemistry and Physics of Lipids

105

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“…BODIPY 493/503 stock solution (1 mg/ml) was prepared by dissolving 5 mg of BODIPY 493/503 (Molecular Probes) in a 5-ml ethanol aliquot and stored at Ϫ20°C. The defatted BSA/oleic acid (OlAc) medium was prepared as indicated (40).…”

Section: Construction Of Flag Epitope-tagged Caveolin-1 Expressionmentioning

confidence: 99%

The Role of Proline in the Membrane Re-entrant Helix of Caveolin-1

Aoki

Thomas

Decaffmeyer

et al. 2010

Journal of Biological Chemistry

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Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro 110 residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the N terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the N terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells, the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the N terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation, and lipid droplet formulation. In addition, we find that this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent-resistant membranes of these cells. Thus, the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.Caveolae are specialized domains of the plasma membrane found in most cell types and particularly abundant in highly differentiated cells, such as endothelial cells, adipocytes, or muscle cells. They were described as invaginations of the plasma membrane (1, 2). Caveolae appear to have a number of functions, including roles in signal transduction, lipid exchange, cell entry, and intracellular delivery of bacterial toxins, viruses, and growth factors (3-13), but the molecular aspects of their formation and functions are still being unraveled (14). Electron microscopy allows the visualization of caveolae as 50 -100-nm flask-shaped invaginations of the plasma membrane or as circularized single or clustered vesicles underneath the plasma membrane. Caveolae are specialized membrane microdomains enriched in sphingolipids, cholesterol, and receptor proteins. It is also the site of NO production and of cholesterol efflux from the cell. Several isoforms of caveolin are characteristic proteins of caveolae (15, 16).Caveolin inserts into membranes of phosphatidylcholine in a cholesterol-dependent manner (17) and is anchored to the membrane with a hydrophobic segment comprising residues 105-125 as well as with three palmitoyl chains attached to Cys residues. Palmitoylated proteins are known to tra...

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Fluorescent Detection of Lipid Droplets and Associated Proteins

Cited by 176 publications

References 28 publications

Group IVA Phospholipase A2 Is Necessary for the Biogenesis of Lipid Droplets

Group IVA Phospholipase A2 Is Necessary for the Biogenesis of Lipid Droplets

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

The Role of Proline in the Membrane Re-entrant Helix of Caveolin-1

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