Direct Membrane Penetration of Oligoarginines by Fluorescence and Cryo-electron Microscopy Combined with Molecular Simulations

Arginine-rich peptides are short amino acid chains capable of spontaneously crossing cellular membranes, with great potential for drug or other cargo delivery. Yet, the mechanisms underlying their cellular penetration are not fully understood. Here, we investigate the modes of action of nonaarginine (R ₉ ) across membranes of increasing compositional and biological complexity. We combine computational, fluorescence microscopy, and cryo-EM approaches to both visualize the membrane structural changes arising from peptide-lipid interactions and provide a molecular rationale for the observed effects. In large unilamellar vesicles, R ₉ binds preferentially to anionic and PE-rich membranes, induces lipid reorganization, and drives pronounced remodelling, including budding, bifurcations, and time-dependent formation of multilamellar stacks. In cell-derived extracellular vesicles, R ₉ -induced remodelling is largely confined to bilamellar bifurcations. In live cells, fluorescent R ₉ forms surface puncta that precede cytosolic entry. Correlative cryo-fluorescence and electron tomography reveals that these puncta correspond to strongly folded, multilamellar membrane structures. We propose that these seemingly contrasting observations can be reconciled within a single R ₉ mechanism of action, involving membrane folding and stacking, where the different observed morphologies arise from the size of the accessible membrane reservoir.