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

Abstract

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.

Keywords: cell-penetrating peptide; electron microscopy; fluorescence microscopy; membrane fusion; molecular dynamics.

Fig. 1.

Fluorescence spectroscopy results. (Top Left and Top Center) Threshold concentrations for leakage induced by ${\mathrm{R}}_9$, ${\mathrm{K}}_9$, and ${\mathrm{R}}_4$ given as inverse of the peptide/lipid ratios for two lipid compositions: DOPE/DOPS 80/20 (lipid 3) and DOPE/DOPC/DOPS 60/20/20 (lipid 4) (the higher the threshold value, the more efficient the peptide is in leaking the vesicles). (Top Right) DLS measurements showing particle growth (right axis, solid circles) overlaid with leakage kinetics (left axis, lines) for ${\mathrm{R}}_9$ for composition and absence of particle growth and leakage for ${\mathrm{R}}_4$. (Bottom) Fluorescence microscopy images showing the effect of ${\mathrm{R}}_9$ on GUV with composition . From Left to Right: 1, no peptide added; 2, shortly after addition of ${\mathrm{R}}_9$; and 3, final state after 1 h. (Scale bars, 50 $\mathrm{\mu }$m.)

Fig. 2.

The schematic mechanisms of ${\mathrm{R}}_9$- and Ca²⁺-mediated vesicle fusion. (A and B) Fusion of different vesicles (in blue and gray) (A), by interface contact (B). (C and D) Adsorption of the charged particles (${\mathrm{R}}_9$ in green and Ca²⁺ in yellow). (E and F) Agglomeration of the bilayers induced by cross-linking. (G) Stalk formation. (H) Opening of the fusion pore. (I and J) ${\mathrm{R}}_9$ translocation via self-fusion of a single vesicle, (K–V) starting from a flat vesicle surface bilayer. (K) Strong adsorption of ${\mathrm{R}}_9$. (K and L) Membrane bifurcation through adhesion and curvature. (M–P) Extension of the bifurcated bilayer (M and N) through ${\mathrm{R}}_9$ cross-linking (O and P). (Q and R) Agglomeration of the bilayers induced by cross-linking of two bilayers on the same vesicle. (S–V) Stalk formation (S and T) and opening of the fusion pore through which additional ${\mathrm{R}}_9$ peptides enter (U and V).

Fig. 3.

Electron micrographs of LUVs in the presence of ${\mathrm{R}}_9$. (A) Vesicles treated with ${\mathrm{R}}_9$ (>60 s) fuse with each other and exhibit bifurcated, multilamellar membranes. (Scale bar, 100 nm.) (B) Example of a multilamellar membrane. (Scale bar, 50 nm.) The violet box is analyzed in D. (C) Example of a membrane bifurcation. The membranes before and after the bifurcation site are analyzed by line scans. The line-scan areas are marked with colored boxes. (D) The histograms are boxed in the same color as the respective line-scan areas in B and C. (D, Top Left) The histogram corresponding to the multilamellar membrane (shown in B) exhibits seven distinct minima attributed to individual membranes.

Fig. 4.

EM and fluorescence microscopy images of the same spot on a fixated HeLa cell in the presence of OG-${\mathrm{R}}_9$. (A) A fluorescence microscopy image of the multilamellar spot showing the presence of the labeled peptide. (B–D) An EM image at three zoom-ins exhibiting bifurcated, multilamellar membranes and vesicle budding. (C) An example of a multilamellar membrane structure. (D) Focus on a budding protrusion.

Fig. 5.

(A) Schematic drawing of vesicle fusion–lipid cross-linking, stalk initialization, and subsequent onset of stalk formation through lipid flip-flop. (B) Time evolution of the Ca²⁺ fusing bilayer system. (C) Time evolution of the same system with ${\mathrm{R}}_9$. (C, Left to Right) Cross-section of systems undergoing fusion: cross-linking, flip-flop, fusion stalk, and fusion pore. (D) Driving forces of the mechanism. Peptide “angling” cross-links vesicles and aggregates membranes, peptide agglomeration and lipid demixing create fusable interface, and negative curvature is generated through strong binding to headgroups.