Journal of Lipid Research
Volume 57, Issue 2, February 2016, Pages 299-309
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Research Articles
A comparative study on fluorescent cholesterol analogs as versatile cellular reporters[S]

https://doi.org/10.1194/jlr.M065326Get rights and content
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Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of Chol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction, and trafficking in cells; hence, it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, specifically subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent Chol analogs in visualizing cellular Chol dynamics.

cholesterol/trafficking
fluorescence microscopy
cholesterol/metabolism
lipid rafts
Niemann-Pick type C disease
membranes/model
giant unilamellar vesicles
giant plasma membrane vesicles
fluorescence correlation spectroscopy
stimulated emission depletion

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This work was supported by funding from the Wolfson Foundation, the Medical Research Council (MRC, Grants MC_UU_12010/unit programmes G0902418 and MC_UU_12025), MRC/BBSRC/ESPRC (Grant MR/K01577X/1), and the Wellcome Trust (Grant 104924/14/Z/14). E.S. was supported by EMBO Long Term and Marie Curie Intra-European Fellowships (MEMBRANE DYNAMICS). M.P.C. was supported by the Alfred Benzon Foundation research stipend. D.W. acknowledges support from the Villum and Novo Nordisk Foundation. A.C. and F.P. acknowledge the European Union Seventh Framework Programme (FP7 2007–2013, “Sphingonet”, grant agreement 289278). F.P. is a Royal Society Wolfson Research Merit Award holder.

    Abbreviations:

    Chol

    cholesterol

    cpm

    counts per molecule

    CTL

    cholestatrienol

    CW

    continuous-wave

    DChol

    6-dansyl-cholesterol

    DHE

    dehydroergosterol

    DOPC

    1,2-dioleoyl-sn-glycero-3-phosphocholine

    ERC

    endocytic recycling compartment

    FCS

    fluorescence correlation spectroscopy

    GPMV

    giant plasma membrane vesicle

    GUV

    giant unilamellar vesicle

    Ld

    liquid disordered

    Lo

    liquid ordered

    %Lo

    liquid ordered partitioning coefficient

    NBD

    7-nitrobenz-2-oxa-1,3-diazol-4-yl

    NPC

    Niemann-Pick type C

    PCC

    Pearson correlation coefficient

    SLB

    supported lipid bilayer

    STED

    stimulated emission depletion

    TF

    TopFluor

    TF-Chol

    TopFluor cholesterol

[S]

The online version of this article (available at http://www.jlr.org) contains a supplement.