Issue 18, 2013

FRET-based imaging of transbilayer movement of pepducin in living cells by novel intracellular bioreductively activatable fluorescent probes

Abstract

To elucidate the mechanisms of direct transmembrane penetration of pepducins, which are artificial lipopeptide G protein-coupled receptor (GPCR) modulators, we developed two types of FRET-based probes, Pep13-FL-SS-Dab (13) targeting the inner leaflet of the lipid bilayer and Pep13-Dab-SS-FL (14) targeting the cytosol, respectively. They are composed of a pepducin moiety and a fluorescent switch component consisting of 5(6)-carboxyfluorescein (FAM) as a fluorophore and dabcyl as a quencher connected through disulfide bond linkage. When they are internalized into the cytosol, intracellular glutathione can cleave the disulfide bond to release the quencher, which results in a turn-on fluorescence signal. Using these probes, we performed live cell imaging of transbilayer movements of pepducins on MCF-7 cells for the first time. The results suggested that the lipid moiety of the probes facilitated pepducin flipping across and tethering to the membrane. The present study raises the possibility of applying the probe architecture for direct intracellular drug delivery.

Graphical abstract: FRET-based imaging of transbilayer movement of pepducin in living cells by novel intracellular bioreductively activatable fluorescent probes

Supplementary files

Additions and corrections

Article information

Article type
Paper
Submitted
18 Dec 2012
Accepted
13 Mar 2013
First published
13 Mar 2013

Org. Biomol. Chem., 2013,11, 3030-3037

FRET-based imaging of transbilayer movement of pepducin in living cells by novel intracellular bioreductively activatable fluorescent probes

M. Tsuji, S. Ueda, T. Hirayama, K. Okuda, Y. Sakaguchi, A. Isono and H. Nagasawa, Org. Biomol. Chem., 2013, 11, 3030 DOI: 10.1039/C3OB27445D

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