FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells: Plasma membrane change induced by cholesterol depletion

Ostasov, Pavel; Sýkora, Jan; Brejchova, Jana; Olżyńska, Agnieszka; Hof, Martin

doi:10.1016/j.chemphyslip.2013.02.006

Cited by 31 publications

(25 citation statements)

References 39 publications

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“…As shown in Fig 3, no green fluorescence was observed in the untreated cells transfected with the different plasmids, suggesting that these specific antibodies could not pass through the intact plasma membrane into the cells ( Fig 3A). After Triton X-100 treatment [19][20][21], green fluorescence was observed in the cells transfected with pXJ-FLAG-2B, pXJ-2B-HA, or pXJ-2BΔ(aa 110-111), suggesting that Triton X-100 completely permeated the plasma membrane and that the antibodies specifically detected the overexpressed 2B proteins with the corresponding tags ( Fig 3B). However, after digitonin treatment [16,22], green fluorescence was still observed in the cells transfected with pXJ-FLAG-2B or pXJ-2B-HA, but not in the cells transfected with pXJ-2BΔ(aa110-111) ( Fig 3C).…”

Section: Membrane Topology Of the Fmdv 2b Protein In Bhk-21cellsmentioning

confidence: 96%

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

Guo

Sun

et al. 2015

PLoS ONE

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Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca2+ concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.

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Section: Membrane Topology Of the Fmdv 2b Protein In Bhk-21cellsmentioning

confidence: 96%

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

Guo

Sun

et al. 2015

PLoS ONE

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“…To follow the fate of the substrate, we incubated cells of B. megaterium MS941 with the fluorescent cholesterol analog 25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol (25-NBD cholesterol) [ 28 ]. Again, this probe associated with the cells’ PHB granules (Fig.…”

Section: Resultsmentioning

confidence: 99%

Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium

et al. 2015

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BackgroundCholesterol, the precursor of all steroid hormones, is the most abundant steroid in vertebrates and exhibits highly hydrophobic properties, rendering it a difficult substrate for aqueous microbial biotransformations. In the present study, we developed a Bacillus megaterium based whole-cell system that allows the side-chain cleavage of this sterol and investigated the underlying physiological basis of the biocatalysis.ResultsCYP11A1, the side-chain cleaving cytochrome P450, was recombinantly expressed in the Gram-positive soil bacterium B. megaterium combined with the required electron transfer proteins. By applying a mixture of 2-hydroxypropyl-β-cyclodextrin and Quillaja saponin as solubilizing agents, the zoosterols cholesterol and 7-dehydrocholesterol, as well as the phytosterol β-sitosterol could be efficiently converted to pregnenolone or 7-dehydropregnenolone. Fluorescence-microscopic analysis revealed that cholesterol accumulates in the carbon and energy storage-serving poly(3-hydroxybutyrate) (PHB) bodies and that the membrane proteins CYP11A1 and its redox partner adrenodoxin reductase (AdR) are likewise localized to their surrounding phospholipid/protein monolayer. The capacity to store cholesterol was absent in a mutant strain devoid of the PHB-producing polymerase subunit PhaC, resulting in a drastically decreased cholesterol conversion rate, while no effect on the expression of the recombinant proteins could be observed.ConclusionWe established a whole-cell system based on B. megaterium, which enables the conversion of the steroid hormone precursor cholesterol to pregnenolone in substantial quantities. We demonstrate that the microorganism’s PHB granules, aggregates of bioplastic coated with a protein/phospholipid monolayer, are crucial for the high conversion rate by serving as substrate storage. This microbial system opens the way for an industrial conversion of the abundantly available cholesterol to any type of steroid hormones, which represent one of the biggest groups of drugs for the treatment of a wide variety of diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0300-y) contains supplementary material, which is available to authorized users.

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“…22-NBD-cholesterol (22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4yl)Amino)-23,24-Bisnor-5-Cholen-3␤-Ol, Molecular Probes) was used as an environment-sensitive probe that localizes in the membrane's interior. 22-NBD-cholesterol fluorescence intensity increases in response to phase changes of membrane lipids from a raft to nonraft fractions; and its distribution in phosphatidylcholine/cholesterol bilayers is opposite to that of cholesterol (36,45). The spectral properties of 22-NBD-cholesterol are almost independent of the phase state of the bilayer (45).…”

Section: Methodsmentioning

confidence: 96%

“…22-NBD-cholesterol fluorescence intensity increases in response to phase changes of membrane lipids from a raft to nonraft fractions; and its distribution in phosphatidylcholine/cholesterol bilayers is opposite to that of cholesterol (36,45). The spectral properties of 22-NBD-cholesterol are almost independent of the phase state of the bilayer (45). A stock solution of 22-NBD-cholesterol (10 mg/0.1 ml in ethanol) was prepared and used at a final concentration of 0.2 M in physiological saline.…”

Section: Methodsmentioning

confidence: 99%

“…22-NBD-cholesterol strongly associates with lipid-disordered membrane regions, and its fluorescence decreases upon lipid-ordering phase formation (36,45). In the mouse neuromuscular junction, interventions that decrease lipid raft formation (marked with CTxB) strongly increase 22-NBD-cholesterol fluorescence (27).…”

Section: Acute Hindlimb Suspension Modulates Membrane Lipid Distributmentioning

confidence: 99%

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Membrane lipid rafts are disturbed in the response of rat skeletal muscle to short-term disuse

Petrov

Кравцова

Matchkov

et al. 2017

American Journal of Physiology-Cell Physiology

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Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular and cellular mechanisms leading to atrophy are not completely understood. We investigate early molecular events that might play a role in skeletal muscle remodeling during mechanical unloading (disuse). The effects of acute (6-12 h) hindlimb suspension on the soleus muscles from adult rats were examined. The integrity of plasma membrane lipid rafts was tested utilizing cholera toxin B subunit or fluorescent sterols. In addition, resting intracellular Ca level was analyzed. Acute disuse disturbed the plasma membrane lipid-ordered phase throughout the sarcolemma and was more pronounced in junctional membrane regions. Ouabain (1 µM), which specifically inhibits the Na-K-ATPase α2 isozyme in rodent skeletal muscles, produced similar lipid raft changes in control muscles but was ineffective in suspended muscles, which showed an initial loss of α2 Na-K-ATPase activity. Lipid rafts were able to recover with cholesterol supplementation, suggesting that disturbance results from cholesterol loss. Repetitive nerve stimulation also restores lipid rafts, specifically in the junctional sarcolemma region. Disuse locally lowered the resting intracellular Ca concentration only near the neuromuscular junction of muscle fibers. Our results provide evidence to suggest that the ordering of lipid rafts strongly depends on motor nerve input and may involve interactions with the α2 Na-K-ATPase. Lipid raft disturbance, accompanied by intracellular Ca dysregulation, is among the earliest remodeling events induced by skeletal muscle disuse.

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FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells: Plasma membrane change induced by cholesterol depletion

Cited by 31 publications

References 39 publications

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium

Membrane lipid rafts are disturbed in the response of rat skeletal muscle to short-term disuse

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