Arginine-rich cell-penetrating peptides do not enter cells by directly passing through a lipid membrane; they instead passively enter vesicles and live cells by inducing membrane multilamellarity and fusion. The molecular picture of this penetration mode, which differs qualitatively from the previously proposed direct mechanism, is provided by molecular dynamics simulations. The kinetics of vesicle agglomeration and fusion by an iconic cell-penetrating peptide-nonaarginine-are documented via real-time fluorescence techniques, while the induction of multilamellar phases in vesicles and live cells is demonstrated by a combination of electron and fluorescence microscopies. This concert of experiments and simulations reveals that the identified passive cell penetration mechanism bears analogy to vesicle fusion induced by calcium ions, indicating that the two processes may share a common mechanistic origin.

Department of Chemistry The Hebrew University of Jerusalem Jerusalem 9190401 Israel

Faculty of Pharmacy University of Helsinki Helsinki 00014 Finland

Fritz Haber Research Center The Hebrew University of Jerusalem Jerusalem 9190401 Israel

Imaging Methods Core Facility at Biocev Faculty of Sciences Charles University 242 50 Vestec Czech Republic

Institute of Biophysics and Biophysical Chemistry University of Regensburg D 93040 Regensburg Germany

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences CZ 166 10 Prague 6 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences CZ 166 10 Prague 6 Czech Republic;

Institute of Physical and Theoretical Chemistry University of Regensburg D 93040 Regensburg Germany

J Heyrovský Institute of Physical Chemistry Czech Academy of Sciences 182 23 Prague 8 Czech Republic

Microbiology and Archaea Centre University of Regensburg D 93040 Regensburg Germany

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