How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides

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How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides, Scientific  Reports, 2016 Feb 3 ; 6:20237

 

The subcellular distribution of bioactive molecules is routinely explored by fluorescence microscopy, including confocal laser scanning microscopy (CLSM). CLSM allows studying the subcellular localization of fluorescent molecules within living cells with excellent time (spinning-disk confocal microscopes) or spatial resolution (stimulated emission depletion microscopy, STED). CLSM is intrinsically based on the fluorescence properties of the molecules, requiring their derivatization with a dye or their fusion with a fluorescent protein. Besides the physicochemical perturbation induced by the addition of a fluorophore to the native molecule6, numerous technical pitfalls have been reported.

Cationic cell-penetrating peptides (CPPs) are short arginine-rich sequences, which penetrate cells and facilitate the cellular uptake of cargoes covalently linked or non-covalently associated (the sequences of three cationic arginine-rich CPPs are reported Supplementary Table 1). There is now abundant and consistent evidences obtained by various experimental protocols that CPPs can penetrate via energy-dependent routes (endocytosis) as well as via energy-independent pathways (“direct-translocation”). The question of the penetration mechanisms is fundamental for the development of CPPs as delivery vectors, because the endosomal route might result in endosomal entrapment or lysosomal degradation of the payload. Unraveling the subcellular distributions of a CPP and of its cargo is crucial to understand the mechanism of internalization, and also to select the best system for delivery to the targeted subcellular compartment. Most of the properties of CPPs are described thanks to CLSM images, as they penetrate silently into living cells. CLSM offers an easy readout to map their intracellular localization, which often, if not always, correlates with their internalization pathways.

 

 

Our method addresses a key issue in fluorescence imaging: the relationship between the local intensity of the fluorescence signal and the local concentration of the fluorescent probe. In particular, we demonstrated that the absence of fluorescence might be due to the self-quenching of the probe. To tackle recurring intramolecular self-quenching issues, chemical modifications of fluorescent scaffolds have been reported. These methods have the drawback to require the tedious synthesis of new dyes. To tackle intermolecular self-quenching, we proposed herein a general and reliable method: to substitute in part the fluorescently labeled peptide by its non-fluorescent analogue. Depending on the ratio fluorescent/non-fluorescent peptides, the presence of the peptide is unveiled in different subcellular regions of the cell.

Therefore, the distribution of an exogenous peptide should never be solely deduced from CLSM pictures obtained with 100% fluorescently labeled peptide. Under such experimental conditions, only the weakly to moderately concentrated subcellular regions are visible. It is noteworthy that such pitfall can explain several apparent contradictions in the field of cell-penetrating peptides regarding their internalization mechanisms.

 

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Scientific  Reports, 2016 Feb 3 ; 6:20237

 

Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequence on the interpretation of intracellular localizations from CLSM pictures. We demonstrated that this discrepancy originated from fluorescence self-quenching. The fluorescence was unveiled by a “dilution” protocol, i.e. by varying the ratio fluorescent/non-fluorescent CPP. This strategy allowed us to rank with confidence the subcellular distribution of several CPPs, contributing to the elucidation of the penetration mechanism. More generally, this study proposes a broadly applicable and reliable method to study the subcellular distribution of any fluorescently labeled molecules.

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How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides

 

Jean-Marie Swiecicki, Frédéric Thiebaut, Margherita Di Pisa, Simon Gourdin-Bertin, Julien Tailhades, Christelle Mansuy, Fabienne Burlina, Serge  Chwetzoff, Germain Trugnan, Gérard Chassaing & Solange Lavielle

 

Scientific  Reports, 2016 Feb 3 ; 6:20237

 

doi: 10.1038/srep20237