Nanoscopic remodeling of lipid bilayers by cell-penetrating peptide penetratin

Cell-penetrating peptides (CPPs) such as penetratin are known to traverse lipid membranes, yet the nanoscale structural consequences of their membrane interactions remain incompletely understood. Using atomic force microscopy (AFM), we visualized penetratin-induced remodeling in supported lipid bilayers (SLBs), focusing on discrete POPC bilayer patches whose exposed edges sensitively report early structural changes. In POPC patches, penetratin first accumulated at patch boundaries, forming elevated peripheral rings, and at higher concentrations generated shallow nanoscale pits across the patch interior. Continuous POPC bilayers exhibited a closely parallel pathway-elevated protrusions at 1 μM penetratin and widespread nanoscale pore-like depressions at 2-4 μM-indicating that similar peptide-lipid structures form even without membrane edges. Bilayers containing anionic POPS showed greatly enhanced susceptibility, progressing from peripheral depressions and aggregates to full fragmentation into nanoscale lipid-peptide particles, whereas cholesterol-containing bilayers remained largely resistant, developing only a few isolated deep defects. Our findings reveal an array of penetratin-induced remodeling events shaped by membrane composition and geometry, providing new mechanistic insight into how penetratin modulates membrane structure at the nanoscale.