Live-cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport.

Autor: Modzel M; Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark., Solanko KA; Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark., Szomek M; Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark., Hansen SK; Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark., Dupont A; Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark., Nåbo LJ; Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, DK-5230 Odense, Denmark., Kongsted J; Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, DK-5230 Odense, Denmark., Wüstner D; Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.
Jazyk: angličtina
Zdroj: Journal of microscopy [J Microsc] 2018 Jul; Vol. 271 (1), pp. 36-48. Date of Electronic Publication: 2018 Mar 08.
DOI: 10.1111/jmi.12691
Abstrakt: Analysis of intracellular cholesterol transport by fluorescence microscopy requires suitable fluorescent analogues of cholesterol. Most existing cholesterol analogues contain lipophilic dyes which can compromise the sterol properties in membranes. An alternative strategy is to introduce additional double bonds into the sterol ring system resulting in intrinsic fluorescence, while at the same time keeping the cholesterol-like properties of the analogues. Existing polyene sterols, such as dehydroergosterol (DHE) or cholestatrienol (CTL), however, contain only three double bonds and suffer from low brightness, significant photobleaching and excitation/emission in the ultraviolet region. Thus, special equipment is required to image such sterols. Here, we describe synthesis, characterization and intracellular imaging of new polyene sterols containing four conjugated double bonds in the sterol ring system. We show that such analogues have red-shifted excitation and emission by ∼20 nm compared to DHE or CTL. The red shift was even more pronounced when preventing keto-enol tautomer equilibration by protecting the 3'-hydroxy group with acetate. We show that the latter analogue can be imaged on a conventional wide field microscope with a DAPI/filipin filter cube. The new polyene sterols show reduced photobleaching compared to DHE or CTL allowing for improved deconvolution microscopy of sterol containing cellular membranes.
(© 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.)
Databáze: MEDLINE
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