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

Scientific Reports
Jean-Marie SwiecickiSolange Lavielle

Abstract

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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Jan 12, 2016·Dalton Transactions : an International Journal of Inorganic Chemistry·S HostachyC Policar
Sep 5, 2018·Chembiochem : a European Journal of Chemical Biology·Alejandro Méndez-ArdoyJavier Montenegro
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Dec 29, 2020·Chemistry : a European Journal·Josep GarciaMacarena Sánchez-Navarro
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May 16, 2019·Chemical Reviews·Patrick G DoughertyDehua Pei
Aug 31, 2021·RSC Chemical Biology·Jannik Bruun LarsenThomas Lars Andresen

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Methods Mentioned

BETA
chemical treatment
chemical modifications
confocal microscopy

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