A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

Erdinc Sezgin, Fatma Betul Can, Falk Schneider, Mathias P. Clausen, Silvia Galiani, Tess A. Stanly, Dominic Waithe, Alexandria Colaco, Alf Honigmann, Daniel Wüstner, Frances Platt, Christian Eggeling

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Abstract

Cholesterol 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 cholesterol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently-labeled cholesterol 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 (GUVs) and giant plasma membrane vesicles (GPMVs); 2) cellular trafficking, specifically subcellular localization in Niemann-Pick C (NPC) disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS) and super-resolution STED-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 cholesterol analogs in visualizing cellular cholesterol dynamics.
Original languageEnglish
JournalJournal of Lipid Research
Volume57
Pages (from-to)299-309
ISSN0022-2275
DOIs
Publication statusPublished - 2016

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Cholesterol
Membranes
Assays
Cell membranes
Fluorescence
Cell Membrane
Spectroscopy
Unilamellar Liposomes
Labels
Visualization
Tissue
Lipids
Experiments

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Sezgin, E., Betul Can, F., Schneider, F., Clausen, M. P., Galiani, S., Stanly, T. A., ... Eggeling, C. (2016). A comparative study on fluorescent cholesterol analogs as versatile cellular reporters. Journal of Lipid Research, 57, 299-309. https://doi.org/10.1194/jlr.M065326
Sezgin, Erdinc ; Betul Can, Fatma ; Schneider, Falk ; Clausen, Mathias P. ; Galiani, Silvia ; Stanly, Tess A. ; Waithe, Dominic ; Colaco, Alexandria ; Honigmann, Alf ; Wüstner, Daniel ; Platt, Frances ; Eggeling, Christian . / A comparative study on fluorescent cholesterol analogs as versatile cellular reporters. In: Journal of Lipid Research. 2016 ; Vol. 57. pp. 299-309.
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abstract = "Cholesterol 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 cholesterol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently-labeled cholesterol 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 (GUVs) and giant plasma membrane vesicles (GPMVs); 2) cellular trafficking, specifically subcellular localization in Niemann-Pick C (NPC) disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS) and super-resolution STED-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 cholesterol analogs in visualizing cellular cholesterol dynamics.",
author = "Erdinc Sezgin and {Betul Can}, Fatma and Falk Schneider and Clausen, {Mathias P.} and Silvia Galiani and Stanly, {Tess A.} and Dominic Waithe and Alexandria Colaco and Alf Honigmann and Daniel W{\"u}stner and Frances Platt and Christian Eggeling",
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Sezgin, E, Betul Can, F, Schneider, F, Clausen, MP, Galiani, S, Stanly, TA, Waithe, D, Colaco, A, Honigmann, A, Wüstner, D, Platt, F & Eggeling, C 2016, 'A comparative study on fluorescent cholesterol analogs as versatile cellular reporters', Journal of Lipid Research, vol. 57, pp. 299-309. https://doi.org/10.1194/jlr.M065326

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters. / Sezgin, Erdinc; Betul Can, Fatma ; Schneider, Falk; Clausen, Mathias P.; Galiani, Silvia; Stanly, Tess A.; Waithe, Dominic; Colaco, Alexandria ; Honigmann, Alf; Wüstner, Daniel; Platt, Frances; Eggeling, Christian .

In: Journal of Lipid Research, Vol. 57, 2016, p. 299-309.

Research output: Contribution to journalJournal articleResearchpeer-review

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AU - Sezgin, Erdinc

AU - Betul Can, Fatma

AU - Schneider, Falk

AU - Clausen, Mathias P.

AU - Galiani, Silvia

AU - Stanly, Tess A.

AU - Waithe, Dominic

AU - Colaco, Alexandria

AU - Honigmann, Alf

AU - Wüstner, Daniel

AU - Platt, Frances

AU - Eggeling, Christian

PY - 2016

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AB - Cholesterol 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 cholesterol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently-labeled cholesterol 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 (GUVs) and giant plasma membrane vesicles (GPMVs); 2) cellular trafficking, specifically subcellular localization in Niemann-Pick C (NPC) disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS) and super-resolution STED-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 cholesterol analogs in visualizing cellular cholesterol dynamics.

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DO - 10.1194/jlr.M065326

M3 - Journal article

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VL - 57

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JO - Journal of Lipid Research

JF - Journal of Lipid Research

SN - 0022-2275

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