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

Abstract

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.

Assessing Cellular Uptake of Exogenous Coenzyme Q10 into Human Skin Cells by X-ray Fluorescence Imaging

“…It has to be noted here that the feature of cell nucleus localization is automatically provided in XRF imaging as long as the incident energy is above the K-absorption edge of those elements which are also present in the cell nucleus. Thus, no additional staining is needed, which often alters the kinetics of uptake and distribution and subsequently influences the applicability of final probes [ 40 ]. The comparison of the elemental images of zinc and iodine (representing Q 10 ) for the fine scan shown in Figure 6 c,d once more demonstrates the homogeneous distribution of iodine among the whole cell area, where Q 10 is primarily expected.…”

Timosaponin Aiii Disrupts Morphological Plasticity and Migration of Breast Adenocarcinoma Through Inhibition of Integrin Internalisation

“…Fluorescent labelling is a powerful strategy to perform high-resolution imaging analysis of target molecules. Indeed, uorescent dye-labelled analogues have been used to examine intracellular localisation and tra cking of cholesterols [34]. However, labelling strategies have not been applied to saponins because the biological activities, including cytotoxicity, of saponins are closely associated with their aglycone moieties and the number and structures of monosaccharides in their sugar chains [35].…”

Cholesterol Chip for the Study of Cholesterol–Protein Interactions Using SPR

“…Others used protein biotinylation and streptavidin (SA) precipitation methods to study their interaction [ 9 , 10 , 11 ]. The modification of cholesterol mainly includes radiolabeled cholesterol [ 9 , 10 , 11 , 12 ], fluorescent labelled cholesterol [ 13 ], spin-labelled cholesterol (using nuclear magnetic resonance (NMR) spectra) [ 14 ] and photoreactive cholesterol [ 15 ].…”

“…The modification of cholesterol is less tedious than modifying each of the 250 cholesterol-binding proteins. The modifications of cholesterol, described above [ 9 , 10 , 11 , 12 , 13 , 14 , 15 ], do not support the use of surface plasmon resonance (SPR) to investigate kinetic interactions. While methods such as nuclear magnetic resonance spectroscopy or X-ray crystallography provide molecular-level data on ligand–protein binding, they are material-consuming processes [ 17 , 18 ] and less useful for understanding binding kinetics and thermodynamics.…”