UMR 7203 : Analyse,Interactions Moléculaires et cellulaires

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Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification

Scientific Reports 6, 36938 (2016)


The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

Proteomic comparison of the EWS-FLI1 expressing cells EF with NIH-3T3 and actin remodeling effect of (R/W)9 cell-penetrating peptide


EuPA Open Proteomics 2016;10:1-8


EWS-FLI1 expression in NIH-3T3 fibroblasts has a profound impact on the phenotype, resulting in the cytoskeleton and adhesive capacity disorganization (EF cells). Besides this, (R/W)9, a cell-penetrating peptide (CPP), has an intrinsic actin remodeling activity in EF cells. To evaluate the impact of the oncogenic protein EWS-FLI1 on proteins expression levels, a quantitative comparison of tumoral EF and non-tumoral 3T3 proteomes was performed. Then to see if we could link the EWS-FLI1 oncogenic transformation to the phenotype reversion induced by (R/W)9, (R/W)9 influence on EF cells proteome was assessed. To our knowledge no such “CPPomic” study has been performed before.



Membrane tubulation by cell penetrating peptides

Cell penetrating peptides induce membrane invaginations in cellular membranes. They induce negative membrane curvature by a metabolic energy independent pathway. This pathway also called 'Physical endocytosis' could be a new mechanism of cell penetrating peptide uptake