Abstract
Cell-penetrating peptides like the cationic human immunodeficiency virus-1 trans-acting activator of transcription (TAT) peptide have the capability to traverse cell membranes and to deliver large molecular cargoes into the cellular interior. We used optical sectioning and state-of-the-art single-molecule microscopy to examine the passive membrane permeation of fluorescently labeled TAT peptides across the membranes of giant unilamellar vesicles (GUVs). In GUVs formed by phosphatidylcholine and cholesterol only, no translocation of TAT up to a concentration of 2 microM into the GUVs could be observed. At the same peptide concentration, but with 40 mol % of anionic phosphatidylserine in the membrane, rapid translocation of TAT peptides across the bilayers was detected. Efficient translocation of TAT peptides was observed across GUVs containing 20 mol % of phosphatidylethanolamine, which is known to induce a negative curvature into membranes. We discovered that TAT peptides are not only capable of penetrating membranes directly in a passive manner, but they were also able to form physical pores with sizes in the nanometer range, which could be passed by small dye tracer molecules. Lipid topology and anionic charge of the lipid bilayer are decisive parameters for pore formation.