Fluorescent Sterols for the Study of Cholesterol Trafficking in Living Cells

McIntosh, Avery L.; Huang, Huan; Atshaves, Barbara P.; Storey, Stephan M.; Gallegos, Adalberto M.; Spencer, Thomas A.; Bittman, Robert; Ohno‐Iwashita, Yoshiko; Kier, Ann B.; Schroeder, Friedhelm

doi:10.1002/9783527623099.ch1

Cited by 10 publications

(16 citation statements)

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“…The result for cholesterol was consistent with previously published data ( 41 ). Thus, similar to other fl uorescent sterols ( 15,16,42 ), DChol exhibited a somewhat higher critical concentration for micelle/microcrystal formation in aqueous buffer than exhibited by cholesterol. Nevertheless, DChol, like cholesterol and other fl uorescent sterols, was very poorly soluble in aqueous buffer.…”

Section: Laser Scanning Confocal Microscopy and Multiphoton Laser Scasupporting

confidence: 91%

“…The above results indicated that the spectral properties of the dansyl group present in DChol were greatly affected by polarity and structure of the environment: i) DChol exhibit the smallest Stokes' shift and highest fl uorescence intensity in most hydrophobic environments (100% dioxane or micelle); ii) DChol showed the largest Stokes' shift and lowest intensity in the more polar environment ( ‫ف‬ 40% dioxane); and iii) as the solvent polarity increased to >40% dioxane, DChol showed increased polarization and light scattering, consistent with the low critical micelle/microcrystalline concentrations exhibited by both cholesterol and fl uorescent sterol analogs in aqueous buffers ( 15,16,42 ).…”

Section: Laser Scanning Confocal Microscopy and Multiphoton Laser Scamentioning

confidence: 61%

“…While the naturally occurring fl uorescent dehydroergosterol (DHE) mimics the behavior of cholesterol, especially in codistributing with cholesterol in purifi ed cholesterolrich and -poor microdomains isolated from plasma membranes, DHE unfortunately has low quantum yield, is easily bleached, absorbs in the UV (i.e., near 325 nm), requires multiphoton excitation for imaging in living cells, and is not sensitive to the polarity of its environment ( 6,(14)(15)(16). Other fl uorescent sterols such as 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3 ␤ -ol (NBD)-cholesterol distribute poorly into membranes and preferentially localize in lipid droplets within the cytoplasm (14)(15)(16).…”

Section: Affi Nity Chromatography Of Purifi Ed Plasma Membranes To Rementioning

confidence: 99%

“…Resolution of confocal imaging is >2,000 Å and some microdomains may be much smaller ( 3,4,6,14,16,38,49 ). Therefore, FRET experiments were performed to determine if DChol and the membrane probes were in proximity to allow fl uorescence energy transfer.…”

Section: Fret To Determine Intermolecular Distance Between Dchol and mentioning

confidence: 99%

See 3 more Smart Citations

Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells

Huang

McIntosh

Atshaves

et al. 2010

Journal of Lipid Research

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Section: Laser Scanning Confocal Microscopy and Multiphoton Laser Scasupporting

confidence: 91%

Section: Laser Scanning Confocal Microscopy and Multiphoton Laser Scamentioning

confidence: 61%

Section: Affi Nity Chromatography Of Purifi Ed Plasma Membranes To Rementioning

confidence: 99%

Section: Fret To Determine Intermolecular Distance Between Dchol and mentioning

confidence: 99%

See 2 more Smart Citations

Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells

Huang

McIntosh

Atshaves

et al. 2010

Journal of Lipid Research

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“…Since cholesterol itself has very few structural/chemical features suitable for UV/VIS or optical imaging, the advent of DHE with many properties mimicking or very similar to those of cholesterol, has proven to be a major asset for probing/elucidating real-time sterol dynamics by video and MPLSM imaging in living cells. For additional insights, the reader is referred to earlier reviews on DHE, synthetic fluorescent sterols, spin labeled sterols, photoreactive sterols, and chemical tags of cholesterol (65)(66)(67)(68).…”

Section: Advent Of Fluorescent Sterolsmentioning

confidence: 99%

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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Changes to cholesterol trafficking in macrophages by Leishmania parasites infection

et al. 2017

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Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse‐chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low‐density lipoproteins indicated that retention of this source of cholesterol is increased in parasite‐containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann–Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites’ membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA‐encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol‐dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses.

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Fluorescent Sterols for the Study of Cholesterol Trafficking in Living Cells

Cited by 10 publications

References 203 publications

Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells

Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Changes to cholesterol trafficking in macrophages by Leishmania parasites infection

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