Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells

Daniel Wüstner, Frederik Wendelboe Lund, Clemens Röhrl, Herbert Stangl

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies.
Original languageEnglish
JournalChemistry and Physics of Lipids
Volume194
Pages (from-to)12-28
ISSN0009-3084
DOIs
Publication statusPublished - 2016

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Cholesterol
Cells
Fluorescence
Sterols
Cell membranes
Photobleaching
Cell Membrane
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
Fluorescence Recovery After Photobleaching
Imaging techniques
Electron microscopy
Lipoproteins
Optical microscopy
Recycling
Microscopy
Electron Microscopy
Coloring Agents
Spectroscopy
Membranes
Lipids

Cite this

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title = "Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells",
abstract = "Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies.",
author = "Daniel W{\"u}stner and Lund, {Frederik Wendelboe} and Clemens R{\"o}hrl and Herbert Stangl",
year = "2016",
doi = "10.1016/j.chemphyslip.2015.08.007",
language = "English",
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Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells. / Wüstner, Daniel; Lund, Frederik Wendelboe; Röhrl, Clemens; Stangl, Herbert.

In: Chemistry and Physics of Lipids, Vol. 194, 2016, p. 12-28.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells

AU - Wüstner, Daniel

AU - Lund, Frederik Wendelboe

AU - Röhrl, Clemens

AU - Stangl, Herbert

PY - 2016

Y1 - 2016

N2 - Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies.

AB - Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies.

U2 - 10.1016/j.chemphyslip.2015.08.007

DO - 10.1016/j.chemphyslip.2015.08.007

M3 - Journal article

C2 - 26291493

VL - 194

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JO - Chemistry and Physics of Lipids

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