Exploring Vesicle Penetration by a Fluorescence Leakage Assay.

To explore the potential connection between cell penetration and membrane fusion, we start by investigating the abilities of R9 as an archetypal CPP, in contrast to non-CPPs like tetraarginine (R4) or nonalysine (26) (K9), to penetrate and cause leakage of large unilamellar vesicles (LUVs) of varying lipid compositions using a fluorescence leakage assay (for details see Materials and Methods and SI Appendix). While, contrary to some published data (12), pure POPC LUVs do not show leakage upon exposure to R9 even at high peptide concentrations, LUVs composed of mixtures of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1,2-dioleoyl-phosphatidylserine (DOPS) exhibit leakage as long as the content of DOPE is sufficiently high (SI Appendix, Table S3). Despite different lipid composition of the present vesicles compared with live cells, we did recover the arginine “magic,” i.e., the high activity of CPPs with more than about 7 amino acids and high arginine content (27). In these vesicles, similar to experiments in cells, R9 was always found to be a more efficient leakage agent than K9 and the essentially inactive R4 peptide (Fig. 1, Top Left). Using dynamic light scattering (DLS) measurements we showed that leakage is accompanied with vesicle aggregation, as the leakage kinetics mimic the increase in time of the mean diameter of the aggregates (see Fig. 1, Top Right and SI Appendix, Table S3 for the DOPE-rich lipid composition).

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Fig. 1.

Fluorescence spectroscopy results. (Top Left and Top Center) Threshold concentrations for leakage induced by R9, K9, and R4 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 R9 for composition 3 and absence of particle growth and leakage for R4. (Bottom) Fluorescence microscopy images showing the effect of R9 on GUV with composition 4. From Left to Right: 1, no peptide added; 2, shortly after addition of R9; and 3, final state after 1 h. (Scale bars, 50 μm.)

Membrane Fusion Induced by Calcium As Well As by Cationic Cell-Penetrating Peptides.

The range of lipid compositions of vesicles capable of being leaked by R9 is at odds with simulations of direct translocation, where a far higher translocation free energy has been predicted for DOPE-rich bilayers than for those rich in POPC (28). However, it seems to match compositions known to enhance vesicle fusion by calcium (20, 21, 29). Both phosphatidylethanolamine (PE) and phosphatidylserine (PS) (as well as several other anionic lipids) are fusogenic in presence of Ca²⁺ (30⇓–32). To verify this correlation, we repeated the experiments with Ca²⁺ instead of R9. Indeed, we were able to observe calcium-induced leakage for lipid compositions that were most susceptible to leakage by the CPPs as well (SI Appendix, Table S3). To obtain a comparable effect, however, the concentration of Ca²⁺ had to be significantly higher than that of R9, even when taking into account the significantly larger charge carried by the latter species.

At high peptide content, the LUV leakage kinetics are described quantitatively by a second-order rate law in the vesicle concentration (for details see SI Appendix, Fig. S11 and the kinetic model in SI Appendix). This indicates that aggregation of vesicles and the double bilayer formed during this process is essential for vesicle leakage. It is revealing that for all lipid compositions at which significant leakage occurs the vesicles also exhibit R9-induced fusion, as detected by a near complete lipid mixing in a Förster resonance energy transfer (FRET) assay (SI Appendix and SI Appendix, Fig. S11). Moreover, the leaky lipid compositions are those known to be susceptible to Ca²⁺-mediated fusion. Note that leaky fusion has been observed previously in conjunction with CPPs and has even been used to classify them (25). In addition to the above circumstantial evidence for a direct connection between cell penetration and membrane fusion we add further experimental support using confocal microscopy, finding agglomeration of giant unilamellar vesicles (GUVs) together with leakage (Fig. 1). We were able to observe fusion of GUVs directly (SI Appendix, Movie S1) and, using Oregon Green 488 (OG)-labeled peptides, we confirmed a preferential adsorption of fluorescently labeled R9 to the vesicles (SI Appendix, Fig. S8). Finally, adding Ca²⁺ instead of R9 to the GUVs, we found a functionally analogous behavior (SI Appendix, Fig. S8).

Ideal Fusion Topologically Precludes Cell Penetration.

The similarities in aggregation/fusion caused by R9 and Ca²⁺ are illustrated in Fig. 2 A–J. In the context of the present study, it is important to note that there is no topological way for peptides to enter the vesicles from the outside (or do the reverse) by an ideal fusion process, within which two unilamellar vesicular structures coalesce, as it merely connects the interiors of the two vesicles. A previous electron microscopy study of Ca²⁺-mediated fusion reported content loss along the fusion diaphragm, attributing it to the strain induced by the deformation during agglomeration (33). A close observation of the GUVs in Fig. 1 indeed reveals that they are significantly deformed as the agglomeration creates nearly planar surfaces at the regions of contact, indicating high tension. While tension supports several fusion steps (34), the large surface tension of the agglomerated, flat, and fusing double bilayers can also lead to rupture—depending on the initial tension of the vesicles it may overcome the line tension leading to a membrane pore. However, a small, transient, pore near the fusion stalk can hardly be the main mechanism of penetration of peptides into vesicles since it will rapidly close (35, 36), making it hard for cargo to pass through. Such pores are also associated with positive curvature, as induced by certain amphipathic helices (37), whereas here we stabilize fusion stalk and pore with negative curvature inducing PE lipids (23) and the hydrophilic R9. For a cell membrane to be ruptured at the fusion diaphragm, a large enough membrane fold would have to be generated first. This is possible only for a system without a large surface tension. However, such membranes are unlikely to be ruptured by a fusion event. Therefore, rupture cannot explain continuous leakage into intact vesicles, nor is it a plausible mechanism for cell penetration as cells tightly regulate their inner pressure and membrane tension and are rich in cholesterol, which increases pore line tension. We show below that a solution lies in locally bifurcating the membrane, leading to multilamellar structures. Such a pathway allows the peptides to enter by fusion without having to form transient pores, as shown schematically in Fig. 2 K–V.

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Fig. 2.

The schematic mechanisms of R9- 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 (R9 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) R9 translocation via self-fusion of a single vesicle, (K–V) starting from a flat vesicle surface bilayer. (K) Strong adsorption of R9. (K and L) Membrane bifurcation through adhesion and curvature. (M–P) Extension of the bifurcated bilayer (M and N) through R9 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 R9 peptides enter (U and V).

Induced Multilamellarity as a Solution to the Topological Conundrum Observed by Cryoelectron Microscopy: Seeing Is Believing.

A tendency of GUVs (13) or cells (12) to become multilamellar upon addition of CPPs has been observed recently. To further explore this idea, we first conducted cryoelectron microscopy (cryo-EM) experiments on LUVs. The obtained cryo-EM images indeed reveal formation of multilamellar domains and lipid bilayer bifurcations after the addition of R9 (Fig. 3). Additional time-resolved FRET experiments on fluorescently labeled LUVs reveal presence of interbilayer energy transfer, which provides independent confirmation for the induction of multilamellar lipid structures by R9 (SI Appendix and SI Appendix, Fig. S11). Importantly, Ca²⁺ ions are also able to fuse and collapse vesicles to multilamellar phases due to the ability of Ca²⁺ to bridge phosphates from different bilayers (33). The cryo-EM structures also provide some additional evidence for fusion, as the LUVs in Fig. 3A are many times larger than those found in the initial state (SI Appendix, Fig. S7A).

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Fig. 3.

Electron micrographs of LUVs in the presence of R9. (A) Vesicles treated with R9 (>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.

Multilamellar structures can be formed via folding of a membrane or by stacking of deflated vesicles. Any process based on direct membrane stacking would, however, add an even number of bilayers in between the vesicles and, therefore, would not lead to leakage via fusion. It is thus a key finding that by counting the lipid bilayers we frequently find odd numbers (Fig. 3D). Moreover, a close inspection of the EM micrographs provides direct evidence for bilayer bifurcation at multiple positions (see Fig. 3C for an example). We conclude that R9 is indeed capable of inducing multilamellarity by membrane adsorption and bifurcation, rendering a cell penetration mechanism via fusion feasible.

The proposed mechanism shares some similarities with the reverse micelle mechanism, proposed in the literature (38, 39). This mechanism also necessitates a small bifurcation, before the membrane edge is closed by forming the reverse micelle. The reverse micelle has negative curvature on the inside and is, therefore, stabilized by similar interactions to those of the bifurcations. We argue that the membrane edge energy can be compensated through extension of stable cross-linked multilamellar domains as seen in the EM pictures. In SI Appendix we show simulations, which indicate the stabilization of the bifurcation by R9, but not R4 even in the absence of cross-linking. The opening of a reverse micelle removes negative curvature from the system. In contrast to this, the fusion stalk (23) and pore both maintain a negative curvature—a finite bilayer thickness translates negative Gaussian curvature into negative mean curvature, present on both membrane leaflets (40). Thereby the whole mechanism can be driven by the same preferential interaction with R9.

Fluorescence and EM on HeLa Cells.

To directly explore the mechanism behind cellular uptake of CPPs in the absence of endocytosis, we first observed penetration into living human HeLa cells by fluorescence confocal microscopy (SI Appendix, Figs. S9 and S10). HeLa cells incubated with 15 μM OG-labeled R9 peptide for 3 min at 4 °C already exhibited surface fluorescence and, in particular, the presence of highly fluorescent foci (SI Appendix, Fig. S9, arrows). Longer incubation of cells with the peptide increased the number of foci detected on HeLa cells. Cytosolic presence of the peptide was observed as soon as 5 min after addition of the peptide to the cells and the number of penetrated cells increased over prolonged incubation time (even when cells were washed after 3 min to remove free peptide from incubation medium). All cells with cytosolic peptide exhibited at least one to two fluorescent foci on their surfaces. Moreover, these foci were found only in cells with endocytosis blocked by low temperature.

These results suggest that R9 rapidly accumulates in very few places on a cell at low temperatures. Previously, EM on cells revealed that addition of R9 leads to the formation of layered membrane domains (41). Motivated by these observations, we performed EM experiments on HeLa cells with added fluorescently labeled R9. In accord with our observations on vesicles and with previous EM on cells (41) we see regions of protrusions, bifurcations, and multilamellarity by incubating cells with R9 at 4 °C. At these conditions active endocytosis is switched off and only passive cell penetration is operational (Fig. 4B). The branched structures (zoomed-in images in Fig. 4C ) are topologically identical to those suggested in the schematic drawing in Fig. 2M and to the structures observed in the LUVs (Fig. 3). The observed protrusions indicate that a strong curvature-generating interaction is at work, as can be seen in Fig. 4D. Specifically, we interpret the budding spherical protrusions as being due to negative (Gaussian) curvature generation, similar to those occurring in a fusion stalk. By overlaying fluorescence images with EM we clearly see that the changes in the bilayer structure correlate with the location of the fluorescently labeled OG-R9, as can be seen in Fig. 4A. Our EM images on HeLa cells are thus consistent with the above results on vesicles, as well as with previous observations showing that giant plasma membrane vesicles (GPMVs) are susceptible to R9 penetration, but only in the presence of intact membrane proteins (3). Note that in contrast to our leakage experiments on LUVs the local, encapsulated foci will allow only very limited diffusion, thereby limiting potential cell toxicity of the CPPs. Their locality at the cell membrane also points to a yet unknown specific interaction which is not present in the synthetic vesicle systems.

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Fig. 4.

EM and fluorescence microscopy images of the same spot on a fixated HeLa cell in the presence of OG-R9. (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.

Molecular Dynamics Simulations: Atomistic Insights.

To gain atomistic insight into the fusion process and its connection to cell penetration we performed molecular dynamics simulations. Previous studies, based on continuum and coarse-grained models, agree that fusion proceeds via a stalk intermediate (23, 35). The stalk is strongly concave, explaining the observed lipid selectivity toward small (PE) headgroups as these stabilize negative curvature.

Our simulation setups involve strongly positively curved bilayer geometries, intended to lower the barriers for fusion (42, 43). The stress hereby induced in the PE-rich bilayers leads to spontaneous stalk formation in our simulations (see Materials and Methods and SI Appendix for full details). Snapshots from the R9- or Ca²⁺-mediated fusion processes are presented in Fig. 5. We find both Ca²⁺ ions and the charged R9 side chains bind to lipid headgroups, primarily at the negatively charged phosphates. Subsequently, we observe mechanistic similarities in the membrane fusion mediated by Ca²⁺ and R9. The first step in the fusion process is cross-linking, i.e., simultaneous binding of lipids from two membrane bilayers by either Ca²⁺ or R9, with the latter found to be a particularly effective cross-linker. As the ions keep the bilayers in close contact, the lipid tails eventually cross-link, too, in what appears to be the rate-determining step of the whole fusion process. This lipid tail cross-linking occurs within about 0.5 μs. Once a cross-linking lipid tail has flip-flopped into the opposing bilayer, the stalk starts forming within a few nanoseconds (Fig. 5 B and C).

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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 R9. (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.

In Fig. 5D we examine the action of R9 on the membrane in close detail. First, we note the long-range cross-linking capability of R9, which is likely to be responsible for its strongly agglomerating effect on vesicles and for stabilizing the multilamellar structures we find in EM. R9 tends to be only partially adsorbed at the membrane and can thus easily reach across the interface and attach to the second bilayer. It is clear that for such a cross-linking a minimum chain length is necessary, which explains the inefficiency of R4 in this respect (see also the control simulation data in SI Appendix, Fig. S3). In addition, R9 also forms agglomerates when cross-linking the interfaces. It is known that R9 binding to membranes is more cooperative than that of K9 (44), making the former a much more efficient agent of membrane fusion and cell penetration than the latter. The attraction of R9 to negatively charged lipids also leads to a lipid sorting effect, with phosphatidylserine lipids (Fig. 5D, orange) being accumulated next to the R9 agglomerate. Electrostatics-based lipid sorting (involving, e.g., gangliosides) may contribute to the action in cells, membranes of which would otherwise not be sufficiently active. In Fig. 5D, Right we show that R9 is preferentially adsorbed to regions of negative curvature (45) (marked by the color-coded surface), as this allows it to efficiently bind its side chains to lipid headgroups (marked in gold). Aggregation of headgroups increases membrane tension in the headgroup region and exposes hydrophobic patches (visible in all panels of Fig. 5D). The exposure of hydrophobic patches in turn lowers hydration repulsion. Once lipid tails from opposing bilayers are in contact, lipid flip-flop can readily occur, starting thus the fusion cascade. At the surface of the fusion stalk and the pore, a very strongly negative Gaussian curvature and a negative mean curvature occur simultaneously. This stabilizes the actual geometry and, therefore, lowers the barrier for fusion. The induction of negative Gaussian curvature was previously proposed as a characteristic of CPPs (4). Importantly for the present mechanism, we do not observe membrane rupture or water permeation during the fusion process.