Cell-penetrating peptides (CPPs) enter cells by direct translocation across the plasma membrane and by endocytosis. This allows them to carry cargo into cells. Whether CPP escape endosomes is however unclear. A laboratory from the department of biomedical sciences now shows that CPPs are unable to escape endosomes. Endocytosis of CPP is therefore a dead end for them. These results indicate that the bioavailability and activity of CPPs and consequently their usefulness in biology and medicine would benefit from the identification of molecules able to favor CPP endosomal escape.
Marc Serulla, Palapuravan Anees, Ali Hallaj, Evgeniya Trofimenko, Tara Kalia, Yamuna Krishnan, Christian Widmann
Cell-penetrating peptides (CPPs) are short (<30 amino acids), generally cationic, peptides that deliver diverse cargos into cells. CPPs access the cytosol either by direct translocation through the plasma membrane or via endocytosis followed by endosomal escape. Both direct translocation and endosomal escape can occur simultaneously, making it non-trivial to specifically study endosomal escape alone. Here we depolarize the plasma membrane and showed that it inhibits the direct translocation of several CPPs but does not affect their uptake into endosomes. Despite good endocytic uptake many CPPs previously considered to access the cytosol via endosomal escape, failed to access the cytosol once direct translocation was abrogated. Even CPPs designed for enhanced endosomal escape showed negligible endosomal escape into the cytosol. Our data reveal that cytosolic localization of CPPs occurs mainly by direct translocation across the plasma membrane. Cell depolarization represents a simple manipulation to stringently test the endosomal escape capacity of CPPs.