Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport

Maciej Modzel, Frederik W. Lund, Daniel Wüstner*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Abstract

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

Original languageEnglish
Title of host publicationCholesterol Homeostasis : Methods and Protocols
EditorsIngrid C. Gelissen, Andrew J. Brown
PublisherHumana Press
Publication date2017
Pages111-140
ISBN (Print)978-1-4939-6873-2
ISBN (Electronic)978-1-4939-6875-6
DOIs
Publication statusPublished - 2017
SeriesMethods in Molecular Biology
Volume1583
ISSN1064-3745

Fingerprint

Photons
Caveolins
Caveolae
Homeostasis
Fluorescence
Membranes
dehydroergosterol
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

Keywords

  • Cholesterol trafficking
  • Fluorescent sterols
  • Life-cell imaging
  • SpatTrack

Cite this

Modzel, M., Lund, F. W., & Wüstner, D. (2017). Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport. In I. C. Gelissen, & A. J. Brown (Eds.), Cholesterol Homeostasis: Methods and Protocols (pp. 111-140). Humana Press. Methods in Molecular Biology, Vol.. 1583 https://doi.org/10.1007/978-1-4939-6875-6_10
Modzel, Maciej ; Lund, Frederik W. ; Wüstner, Daniel. / Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport. Cholesterol Homeostasis: Methods and Protocols. editor / Ingrid C. Gelissen ; Andrew J. Brown. Humana Press, 2017. pp. 111-140 (Methods in Molecular Biology, Vol. 1583).
@inbook{ee624c7eebe94564841e4992c9eebcd8,
title = "Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport",
abstract = "Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol{\circledR}; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.",
keywords = "Cholesterol trafficking, Fluorescent sterols, Life-cell imaging, SpatTrack",
author = "Maciej Modzel and Lund, {Frederik W.} and Daniel W{\"u}stner",
year = "2017",
doi = "10.1007/978-1-4939-6875-6_10",
language = "English",
isbn = "978-1-4939-6873-2",
series = "Methods in Molecular Biology",
publisher = "Humana Press",
pages = "111--140",
editor = "{C. Gelissen}, Ingrid and {J. Brown}, Andrew",
booktitle = "Cholesterol Homeostasis",
address = "United States",

}

Modzel, M, Lund, FW & Wüstner, D 2017, Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport. in I C. Gelissen & A J. Brown (eds), Cholesterol Homeostasis: Methods and Protocols. Humana Press, Methods in Molecular Biology, vol. 1583, pp. 111-140. https://doi.org/10.1007/978-1-4939-6875-6_10

Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport. / Modzel, Maciej; Lund, Frederik W.; Wüstner, Daniel.

Cholesterol Homeostasis: Methods and Protocols. ed. / Ingrid C. Gelissen; Andrew J. Brown. Humana Press, 2017. p. 111-140 (Methods in Molecular Biology, Vol. 1583).

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

TY - CHAP

T1 - Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport

AU - Modzel, Maciej

AU - Lund, Frederik W.

AU - Wüstner, Daniel

PY - 2017

Y1 - 2017

N2 - Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

AB - Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

KW - Cholesterol trafficking

KW - Fluorescent sterols

KW - Life-cell imaging

KW - SpatTrack

U2 - 10.1007/978-1-4939-6875-6_10

DO - 10.1007/978-1-4939-6875-6_10

M3 - Book chapter

C2 - 28205171

AN - SCOPUS:85013032167

SN - 978-1-4939-6873-2

T3 - Methods in Molecular Biology

SP - 111

EP - 140

BT - Cholesterol Homeostasis

A2 - C. Gelissen, Ingrid

A2 - J. Brown, Andrew

PB - Humana Press

ER -

Modzel M, Lund FW, Wüstner D. Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport. In C. Gelissen I, J. Brown A, editors, Cholesterol Homeostasis: Methods and Protocols. Humana Press. 2017. p. 111-140. (Methods in Molecular Biology, Vol. 1583). https://doi.org/10.1007/978-1-4939-6875-6_10