Mechanism of Cell Penetration by Permeabilization of Late Endosomes: Interplay between a Multivalent TAT-Like Cell-Penetrating Peptide and the Lipid Bis(Monoacylglycerol)Phosphate

Many cellular delivery reagents enter the cytosolic space of cells by escaping the lumen of endocytic organelles and, more specifically, late endosomes. The mechanisms involved in endosomal membrane permeation remain largely unresolved, which impedes the improvement of delivery agents. Herein, we investigate how 3TAT, a branched analog of the cell-penetrating peptide (CPP) TAT, achieves the permeabilization of bilayers containing bis(monoacylglycerol)phosphate (BMP), a lipid found in late endosomes. We establish that the peptide does not induce the leakage of individual lipid bilayers. Instead, leakage requires contact between membranes. Peptide-driven bilayer contacts lead to fusion, lipid mixing, and, critically, peptide encapsulation within proximal bilayers. Notably, this encapsulation is a distinctive property of BMP that explains the specificity of the CPP’s membrane leakage activity. These results therefore support a novel model of cell penetration that requires both BMP and the vicinity between bilayers, two features unique to BMP-rich and multivesicular late endosomes.