Cholesterol Distribution in Living Cells: Fluorescence Imaging Using Dehydroergosterol as a Fluorescent Cholesterol Analog

Mukherjee, Sushmita; Zha, Xiaohui; Tabas, Ira; Maxfield, Frederick R.

doi:10.1016/s0006-3495(98)77632-5

Cited by 307 publications

(303 citation statements)

References 46 publications

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“…The significance of the above findings in living cells was confirmed with DHE and other fluorescent sterols in a variety of cultured cells including L-cells, CHO cells, macrophages, and hepatocyte derived cell lines (39,43,45,160,(174)(175)(176). Studies with transfected L-cells showed that uptake and efflux of fluorescent sterols was highly dependent on the expression of cholesterol binding proteins (e.g.…”

Section: Physiological Significance Of In Vitro Studies With Dhe and mentioning

confidence: 66%

“…Recent improvements in imaging microscopy allowed the direct, real-time visualization of DHE in living cells by conventional (DHE delivered as micro-crystals) or video (DHE delivered as cyclodextrin complexes) UV fluorescence microscopy (39,160,(174)(175)(176) and by MPLSM (DHE delivered as LUV or micro-crystals) multiphoton laser scanning microscopy (71,82,203,204). Analysis of intracellular localization and distribution of DHE at the plasma membrane can be accomplished after segmentation of specific regions of interest such as cellular organelles and performed on a cell by cell basis (70,203 stastic-based technique, two small windows are created in the image and the pixel intensities in each window ranked from the lowest to highest; then a comparison is made by using Miller's rank statistic.…”

Section: Plasma Membrane Sterol-rich Microdomains: Real-time Imaging mentioning

confidence: 99%

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Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent of dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular sterol trafficking in living organisms. DHE is a naturally-occurring, fluorescent sterol analog that faithfully mimics many of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins, is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock solution to cell culture media is effective, but this process forms an aqueous dispersion of dehydroergosterol crystals which can result in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques can deliver large quantities of monomeric dehydroergosterol with significant distribution into the plasma membrane. The properties and behavior of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging in living cells indicate that this naturally-occurring fluorescent sterol is a useful mimic for probing the properties of cholesterol in these systems. Keywordsdehydroergosterol; cholesterol; ergosterol; fluorescent sterols; microscopy Historical PerspectiveIn order to resolve the dynamics and factors governing cholesterol trafficking within cells, it is important to recognize that the cholesterol molecule has very few polar constituents (Fig. 1A). Consequently, cholesterol is poorly soluble in aqueous environments such as cytosol as evidenced by critical micellar concentrations of cholesterol and other sterols being very low, *To whom correspondence should be addressed: Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 862-1433, FAX: (979) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript near 20-30 nM (1-5). The physiological impact of cholesterol's low aqueous solubility is that spontaneous cholesterol desorption from the cytofacial leaflet of the plasma membrane or lysosomal membrane into the cytosol for transfer to intracellular sites for esterification, oxidation, or secretion is extremely slow (t 1/2 3 hours-days) (6-9). Therefore, it is important to resolve factors...

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Section: Physiological Significance Of In Vitro Studies With Dhe and mentioning

confidence: 66%

Section: Plasma Membrane Sterol-rich Microdomains: Real-time Imaging mentioning

confidence: 99%

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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“…Increased bulk as well as where the fluorophore is positioned could strongly influence a lipid's binding properties. As recently pointed out for cholesterol [40,41], NBD derivatization could also affect how a lipid analyte is distributed within cells. Based on these considerations, the desirability of employing unmodified lipids for binding or transfer-distribution studies is obvious.…”

Section: Discussionmentioning

confidence: 92%

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Kernstock

Girotti

2007

Analytical Biochemistry

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A new approach for analyzing lipid-lipid transfer protein interactions is described. The transfer protein is genetically engineered for expression with a C-terminal biotinylated peptide extension (AviTag ® ). This allows protein anchoring to a streptavidin-coated chip for surface plasmon resonance (SPR)-based assessment of lipid binding. Sterol carrier protein-2 (SCP-2), involved in the intracellular trafficking of cholesterol, fatty acids and other lipids, was selected as the prototype. Biotinylated SCP-2 (bSCP-2) was expressed in E. coli, purified to homogeneity by mutated streptavidin (SoftLink ® ) affinity chromatography, and confirmed by mass spectrometry to contain one biotin group at the expected position. Intermembrane [ 14 C]cholesterol transfer was strongly enhanced by bSCP-2, demonstrating that it was functional. Using bSCP-2 immobilized on a Biacore streptavidin chip, we determined on-and off-rate constants along with equilibrium dissociation constants for the following analytes: oleic acid, linoleic acid, cholesterol, and fluorophore (NBD)-derivatized cholesterol. The dissociation constant for NBD-cholesterol was similar to that determined by fluorescence titration for SCP-2 in solution, thus validating the SPR approach. This method can be readily adapted to other transfer proteins and has several advantages over existing techniques for measuring lipid binding, including (i) ability to study lipids in their natural states, i.e. without relatively large reporter groups; and (ii) ability to measure on-and off-rate constants as well as equilibrium constants.

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“…Dehydroergosterol (DHE) is a natural sterol with intrinsic fluorescence properties, which is found in low amounts in S. cerevisiae 22,23 . DHE is very similar to ergosterol in structural and chemical properties with only single double bond difference and it can replace ergosterol in yeast without any functional interference 24 . To test whether Upc2 directly binds to yeast sterols, we performed the fluorescent DHE-binding assay using purified CTDs of recombinant Upc2 (residues 598-913).…”

Section: Resultsmentioning

confidence: 99%

Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2

et al. 2015

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Transcriptional regulation of ergosterol biosynthesis in fungi is crucial for sterol homeostasis and for resistance to azole drugs. In Saccharomyces cerevisiae, the Upc2 transcription factor activates the expression of related genes in response to sterol depletion by poorly understood mechanisms. We have determined the structure of the C-terminal domain (CTD) of Upc2, which displays a novel a-helical fold with a deep hydrophobic pocket. We discovered that the conserved CTD is a ligand-binding domain and senses the ergosterol level in the cell. Ergosterol binding represses its transcription activity, while dissociation of the ligand leads to relocalization of Upc2 from cytosol to nucleus for transcriptional activation. The C-terminal activation loop is essential for ligand binding and for transcriptional regulation. Our findings highlight that Upc2 represents a novel class of fungal zinc cluster transcription factors, which can serve as a target for the developments of antifungal therapeutics.

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Cholesterol Distribution in Living Cells: Fluorescence Imaging Using Dehydroergosterol as a Fluorescent Cholesterol Analog

Cited by 307 publications

References 46 publications

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2

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