title: A comparative study on fluorescent cholesterol analogs as versatile cellular reporters.
abstract: 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.
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX39DS Oxford, United Kingdom erdinc.sezgin@rdm.ox.ac.uk christian.eggeling@rdm.ox.ac.uk.
authors.forename: Erdinc
authors.initials: E
authors.lastname: Sezgin
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX39DS Oxford, United Kingdom.
authors.forename: Fatma Betul
authors.initials: FB
authors.lastname: Can
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX39DS Oxford, United Kingdom.
authors.forename: Falk
authors.initials: F
authors.lastname: Schneider
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX39DS Oxford, United Kingdom MEMPHYS-Center for Biomembrane Physics, Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Mathias P
authors.initials: MP
authors.lastname: Clausen
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX39DS Oxford, United Kingdom.
authors.forename: Silvia
authors.initials: S
authors.lastname: Galiani
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX39DS Oxford, United Kingdom.
authors.forename: Tess A
authors.initials: TA
authors.lastname: Stanly
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX39DS Oxford, United Kingdom.
authors.forename: Dominic
authors.initials: D
authors.lastname: Waithe
authors.affiliation: Department of Pharmacology, University of Oxford, OX13QT Oxford, United Kingdom.
authors.forename: Alexandria
authors.initials: A
authors.lastname: Colaco
authors.affiliation: Max Planck Institute of Cell Biology and Genetics, 01307 Dresden, Germany.
authors.forename: Alf
authors.initials: A
authors.lastname: Honigmann
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wüstner
authors.affiliation: Department of Pharmacology, University of Oxford, OX13QT Oxford, United Kingdom.
authors.forename: Frances
authors.initials: F
authors.lastname: Platt
authors.affiliation: MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX39DS Oxford, United Kingdom erdinc.sezgin@rdm.ox.ac.uk christian.eggeling@rdm.ox.ac.uk.
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
mesh_terms: D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002784:Cholesterol / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D053835:Unilamellar Liposomes / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D053835:Unilamellar Liposomes; D002784:Cholesterol
keywords: Niemann-Pick type C disease; cholesterol/metabolism; cholesterol/trafficking; fluorescence correlation spectroscopy; fluorescence microscopy; giant plasma membrane vesicles; giant unilamellar vesicles; lipid rafts; membranes/model; stimulated emission depletion