Influence of fluorophore and linker length on the localization and trafficking of fluorescent sterol probes

Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore...

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Bibliographic Details
Published in:Scientific reports 2020-12, Vol.10 (1), p.22053-22053, Article 22053
Main Authors: Králová, Jarmila, Jurášek, Michal, Mikšátková, Lucie, Marešová, Anna, Fähnrich, Jan, Cihlářová, Petra, Drašar, Pavel, Bartůněk, Petr, Král, Vladimír
Format: Article
Language:English
Subjects:
631/45
631/80
631/92
Cell permeability
Cholesterol
Fluorescent indicators
Fluorophores
Humanities and Social Sciences
Intracellular
Kinetics
Labeling
Localization
multidisciplinary
Npc1 protein
Physicochemical properties
Probes
Protein transport
Science
Science (multidisciplinary)
Sterols
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Summary:Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore, affect the localization and trafficking properties of fluorescent sterol probes. Varying lengths of linker using the same fluorophore affected cell penetration and retention in specific cell compartments. A C-4 linker was confirmed as optimal. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions also showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. These probes also strongly labelled endo/lysosomal compartment in cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, that overlapped with filipin staining of cholesterol. Overall, the present study demonstrates that the physicochemical properties of the fluorophore/linker pairing determine the kinetics of uptake and distribution and subsequently influence the applicability of final probes.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-020-78085-9