Arginine-rich cell-penetrating peptides induce membrane multilamellarity and subsequently enter via formation of a fusion pore.

Liposome Experiments. Leakage. Calcein-containing vesicles were stirred at room temperature with LUV buffer in a quartz cuvette to obtain 1.5 mL of solution. The calcein fluorescence was monitored at 520 nm, with excitation at 495 nm. After an initial stirring phase of no less than 200 s, 3–6 μ L peptide in buffer solution was added. After the fluorescence intensity reached a plateau, 50 μ L of TRITON-X was added. Fluorescence intensity measurements were performed on a Fluorolog-3 spectrofluorimeter (model FL3–11; JobinYvon Inc.) equipped with a xenon-arc lamp. See SI Appendix for further details. Confocal microscopy. GUVs labeled with DiD were prepared for confocal microscopy using electroformation in a 300-mOsm/L sucrose solution. Prepared GUVs were diluted with a glucose buffer [9 mM HEPES, pH 7.40 (KOH), 90 mM KCl, 90 mM EDTA, 120 mM glucose, 300 mOsm/L, filtrated] with 20 μ L of 50 nM Atto 488 to a total volume of 300 μ L. Images were recorded using an Olympus IX81 laser scanning confocal microscope. For further details see SI Appendix . Cryo-EM. For cryo-EM sample preparation, 4 μ L of the sample was applied to plasma-cleaned EM grids [400-mesh copper grids, covered with Quantifoil film (R1.2/1.3)]. Samples were plunge frozen on the grids in liquid ethane in a Grid Plunger (Leica EM GP; Leica Microsystems GmbH) with the following parameters: preblotting exposure 5 s, blotting time 1.7 s, no postblotting exposure. Chamber humidity was set to 95% at 22 °C. The LUV solution was treated with R 9 (c = 25 mM) in a ratio of 10:1 for t > 60 s immediately before plunge freezing. Cryoelectron micrographs were collected on a JEM-2100F (JEOL Germany GmbH) operated at 200 kV (see SI Appendix for further details). Cell Experiments. Forty thousand HeLa cells were seeded to a well of μ -slide (ibiTreat; ibidi) 16–20 h before the experiment. Cells were washed with SF-DMEM and kept at 4 °C for 15 min to inhibit endocytic processes. For treatment, a precooled (4 °C) 15- μ M solution of a peptide in SF-DMEM was added to cells via media exchange and incubated for indicated periods of time at 4 °C. In selected cases, cells were treated for 3 min with a peptide at 4 °C, washed with precooled SF-DMEM, and further incubated for an indicated period at 4 °C in fresh SF-DMEM. Cells were imaged using a scanning confocal microscope (FluoView 1000; Olympus) and the tomograms were acquired on a Titan Halo transmission electron microscope (see SI Appendix for details). Computational Details. We use all atom molecular dynamics (MD) for the fusion process: In a first setup, we created two curved membranes via lipid population imbalances at the two leaflets of each bilayer. In the second setup, we put a very small vesicle composed of DOPE (80%) and DOPS (20%) in the unit cell and let it fuse with its periodic image. Both of these approaches facilitate formation of the stalk without enforcing its shape. For calcium fusion we used optimized charge-scaled force fields for ions, to account effectively for electronic polarization effects. For vesicle aggregation and bifurcation calculations we used coarse-graining methods. See SI Appendix for full details.